DNA encoding a human 5-HT1E receptor and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid molecule encoding a human 5-HT 1E  receptor, an isolated protein which is a human 5-HT 1E  receptor, vectors comprising an isolated nucleic acid molecule encoding a human 5-HT 1E  receptors, mammalian cells comprising such vectors, antibodies directed to the human 5-HT 1E  receptor, nucleic acid probes useful for detecting nucleic acid encoding human 5-H 1E  receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human 5-HT 1E  receptor, pharmaceutical compounds related to human 5-HT 1E  receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human 5-HT 1E  receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human 5-HT 1E  receptor.

This is a continuation of U.S. application Ser. No. 08/194,113, filedFeb. 8, 1994, abandoned which is a continuation of U.S. application Ser.No. 07/803,626, field Dec. 2, 1991, abandoned the contents of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Since the purification of a pressor substance in blood serum termedserotonin (Rapport et al., 1947) and later identified as5-hydroxytryptamine (5-HT)(Rapport, 1949), there has been a plethora ofreports demonstrating that this indoleamine not only plays a role in thefunctioning of peripheral tissues but, indeed, performs a key role inthe brain as a neurotransmitter. Certainly, the anatomical localizationof serotonin and serotonergic neurons in both the peripheral and centralnervous systems supports its role in such diverse physiologic andbehavioral functions as pain perception, sleep, aggression, sexualactivity, hormone secretion, thermoregulation, motor activity,cardiovascular function, food intake and renal regulation (For reviewsee Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka,1991). Taken together, it appears that serotonin plays an important rolein homeostasis and in modulating responsiveness to environmentalstimuli. Accordingly, studies demonstrating that abnormalities in theserotonergic system may be associated with disease states has created adrug development effort towards agents which may selectively modulatethe function of serotonin (Glennon, 1990).

In relation to the characterization of physiologic or biochemicalresponses resulting from the release of serotonin are simultaneousinvestigations examining the receptor sites responsible for the actionselicited by the indoleamine transmitter. Following early in vitropharmacological assays describing the existence of two differentserotonin receptors, designated as D and M, in the guinea pig ileum(Gaddum and Picarelli, 1957), the advent of receptor binding techniquein the 1970's has brought to light during the last decade the diversityof 5-HT receptors existing in both the brain and peripheral tissues.Thus, although the concept of D and M receptors has not beeninvalidated, serotonin receptors not fitting either category have beenidentified using radioligand methods. To date using this technique,there appears to be four classes of serotonin receptors found in thebrain: 5-HT₁, 5-HT₂, 5-HT₃ and, 5-HT₄ (Peroutka, 1991). Furthermore,5-HT1 sites have been subclassified as: 5-HT_(1A), 5-HT_(1B), 5-HT_(1C),5-HT_(1D) (Hamon et al., 1990) and 5-HT_(1E) (Leonhardt et al., 1989).Although a detailed characterization of the 5-HT_(1E) binding site islacking, extensive pharmacologic, biochemical and functional propertieshave clearly shown that the other four subtypes of 5-HT₁ sites arereceptors according to classical criteria. Interestingly, the 5-HT_(1E)binding site was first observed in human cortical tissue using [³ H]5-HTas the radioligand probe in the presence of 5-carboxyamidotryptamine andmesulergine to mask other members of the 5-HT_(1E) receptor class. Theaffinity constants of the nine drugs tested indicated a uniquepharmacological profile. In particular, the low affinity of 5-CT andergotamine seemed to clearly discriminate the pharmacologically defined5-HT_(1D) site from that of this novel serotonergic site. Importantly,it was demonstrated that 5-HT_(1E) -sites are saturable and exist in adensity consistent with other known neurotransmitter receptors.Furthermore, this site appeared to interact with a GTP-binding protein.Overall, the data provided a framework suggesting that the 5-HT_(1E)binding site may represent a functional receptor.

During the last few years, the field of molecular biology has providedan important facet to receptor research by cloning these proteins andallowing more precise characterizations in isolated systems (Hartig etal., 1990). This has been accomplished for the 5-HT_(1A) (Fargin et al.,1988), 5-HT_(1C) (Julius et al., 1988), 5-HT_(1D) (Branchek et al.,1990) and 5-HT₂ receptors (Pritchett et al., 1988). Thus, there is nodoubt that these binding sites represent "true" functional receptors.Indeed, the pharmacological characterization of serotonin receptorsinvolved in various physiological or biochemical functions is a keycomponent of drug development for the serotonergic system. As one candeduce from the diversity of serotonin binding sites, many targets areavailable for advancement in selective drug design. The coupling ofmolecular biological methods to pharmacological characterizationparticularly for cloned human receptors will open new avenues forpharmaceutical development which have not been previously explored.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid molecule encoding ahuman 5-HT_(1E) receptor.

This invention also provides an isolated protein which is a human5-HT_(1E) receptor.

This invention provides a vector comprising an isolated nucleic acidmolecule encoding a human 5-HT_(1E) receptor.

This invention also provides vectors such as plasmids comprising a DNAmolecule encoding a human 5-HT_(1E) receptor, adapted for expression ina bacterial cell, a yeast cell, or a mammalian cell which additionallycomprise the regulatory elements necessary for expression of the DNA inthe bacterial, yeast, or mammalian cells so located relative to the DNAencoding the 5-HT_(1E) receptor as to permit expression thereof.

This invention provides a mammalian cell comprising a DNA moleculeencoding a human 5-HT_(1E) receptor.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a human 5-HT_(1E) receptor can bind toa human 5-HT_(1E) receptor which comprises contacting a mammalian cellcomprising an isolated DNA molecule encoding a human 5-HT_(1E) receptorwith the ligand under conditions permitting binding of ligands known tobind to a 5-HT_(1E) receptor, detecting the presence of any of theligand bound to a human 5-HT_(1E) receptor, and thereby determiningwhether the ligand binds to a human 5-HT_(1E) receptor.

This invention also provides a method for determining whether a ligandnot known to be capable of binding to the human 5-HT_(1E) receptor canfunctionally activate its activity or prevent the action of a ligandwhich does so. This comprises contacting a mammalian cell comprising anisolated DNA molecule which encodes a human 5-HT_(1E) receptor with theligand under conditions permitting the activation or blockade of afunctional response, detected by means of a bioassay from the mammaliancell such as a second messenger response, and thereby determiningwhether the ligand activates or prevents the activation of the human5-HT_(1E) receptor functional output.

This invention further provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, the human 5-HT_(1E)receptor on the surface of a cell which comprises contacting a mammaliancell comprising an isolated DNA molecule encoding a human 5-HT_(1E)receptor with a plurality of drugs, determining those drugs which bindto the mammalian cell, and thereby identifying drugs which specificallyinteract with, and bind to, a human 5-HT_(1E) receptor.

This invention also provides a method of screening drugs to identifydrugs which interact with, and activate or block the activation of, thehuman 5-HT_(1E) receptor on the surface of a cell which comprisescontacting the mammalian cell comprising an isolated DNA moleculeencoding and expressing a human 5-HT_(1E) receptor with a plurality ofdrugs, determining those drugs which activate or block the activation ofthe receptor in the mammalian cell using a bioassay such as a secondmessenger assays, and thereby identifying drugs which specificallyinteract with, and activate or block the activation of, a human5-HT_(1E) receptor.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1E) receptor.

This invention also provides a method of detecting expression of the5-HT_(1E) receptor on the surface of a cell by detecting the presence ofmRNA coding for a 5-HT_(1E) receptor which comprises obtaining totalmRNA from the cell and contacting the mRNA so obtained with a nucleicacid probe comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of a nucleic acid molecule encoding a human 5-HT_(1E) receptorunder hybridizing conditions, detecting the presence of mRNA hybridizedto the probe, and thereby detecting the expression of the 5-HT_(1E)receptor by the cell.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a human 5-HT_(1E) receptor so as to prevent translation ofthe mRNA molecule.

This invention provides an antibody directed to a human 5-HT_(1E)receptor.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human 5-HT_(1E) receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human 5-HT_(1E)receptor so mutated as to be incapable of normal receptor activity, andnot expressing native 5-HT_(1E) receptor. This invention furtherprovides a transgenic nonhuman mammal whose genome comprises antisenseDNA complementary to DNA encoding a human 5-HT_(1E) receptor so placedas to be transcribed into antisense mRNA which is complementary to mRNAencoding a 5-HT_(1E) receptor and which hybridizes to mRNA encoding a5-HT_(1E) receptor thereby reducing its translation.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1E) receptors whichcomprises producing a transgenic nonhuman animal whose levels of human5-HT_(1E) receptor expression are varied by use of an inducible promoterwhich regulates human 5-HT_(1E) receptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1E) receptors whichcomprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of human 5-HT_(1E) receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the expression of a specific human 5-HT_(1E)receptor allele which comprises: a. obtaining DNA of subjects sufferingfrom the disorder: b. performing a restriction digest of the DNA with apanel of restriction enzymes; c. electrophoretically separating theresulting DNA fragments on a sizing gel; d. contacting the resulting gelwith a nucleic acid probe capable of specifically hybridizing to DNAencoding a human 5-HT_(1E) receptor and labelled with a detectablemarker; e. detecting labelled bands which have hybridized to the DNAencoding a human 5-HT_(1E) receptor labelled with a detectable marker tocreate a unique band pattern specific to the DNA of subjects sufferingfrom the disorder; f. preparing DNA obtained for diagnosis by steps a-e;and g. comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

This invention provides a method of preparing the isolated 5-HT_(1E)receptor which comprises inducing cells to express 5-HT_(1E) receptor,recovering the receptor from the resulting cells and purifying thereceptor so recovered.

This invention also provides a method of preparing the isolated5-HT_(1E) receptor which comprises inserting nucleic acid encoding5-HT_(1E) receptor in a suitable vector, inserting the resulting vectorin a suitable host cell, recovering the receptor produced by theresulting cell, and purifying the receptor so recovered.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a receptor so as to prevent translation of the mRNAmolecule.

This invention also provides a transgenic nonhuman mammal expressing DNAencoding a receptor.

This invention further provides a transgenic nonhuman mammal expressingDNA encoding a receptor so mutated as to be incapable of normal receptoractivity, and not expressing native receptor.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a transgenic nonhuman animal whose levels of receptorexpression are varied by use of an inducible promoter which regulatesreceptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a panel of transgenic nonhuman animals each expressing adifferent amount of the receptor.

This invention further provides a transgenic nonhuman mammal whosegenome comprises antisense DNA complementary to DNA encoding a receptorso placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the receptor and which hybridizes to mRNAencoding the receptor thereby preventing its translation.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a receptor can bind to a receptorwhich comprises contacting a mammalian cell comprising an isolated DNAmolecule encoding the receptor with the ligand under conditionspermitting binding of ligands known to bind to a receptor, detecting thepresence of any of the ligand bound to the receptor, and therebydetermining whether the ligand binds to the receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Nucleotide and deduced amino acid sequence of gene 5-HT_(1E).(Seq. I.D. Nos. 1 and 2).

Numbers above the nucleotide sequence indicate nucleotide position. DNAsequence was determined by the chain termination method of Sanger, etal., on denatured double-stranded plasmid templates using the enzymeSequenase. Deduced amino acid sequence (single letter code) of a longopen reading frame is shown.

FIG. 2. Comparison of the human 5-HT_(1E) receptor primary structureswith other serotonin receptors. (Seq. I.D. Nos. 3 - 5HT_(1A) ; 4-5HT_(1C) ; 5- 5HT_(1D)α ; 6- 5HT_(1D)β ; 7- 5HT₂)

Amino acid sequences (single letter code) are aligned to optimizehomology. The putative transmembrane spanning domains are indicated bystars and identified by Roman numerals (TM I-VII).

FIG. 3. Respective pKi values of various drugs for the inhibition of [³H]5-HT specific binding to the cloned 5-HT_(1E) receptor and humancortical membrane preparations containing the native 5-HT_(1E) receptor.

Values for the native membrane preparation are taken from Leonhardt etal., 1989. "r" is the correlation coefficient between pKi valuescalculated for the two receptor preparations and clearly indicates thesimilarity in binding profile.

FIG. 4. 5-HT concentration-effect curve for the inhibition offorskolin-stimulated CAMP formation in Y1 cells transfected with the5-HT_(1E) receptor.

Values are expressed as a percentage of forskolin(Fsk)-stimulated cAMPproduction.

FIG. 5. 5-HT_(1E) mRNA localization in the human brain.

cDNA was reverse transcribed from total RNA (5 μg) using random hexamers(500 pmoles). One microgram of cDNA was subjected to 30 cycles of PCRamplification using 5'- and 3'-primers directed to the third cytoplasmicloop of the 5HT1E receptor gene. Amplified fragments were subjected toSouthern blot analysis using an end-labeled oligonucleotide probe whichwas internal to the PCR primers. (+) control consisted of 5HT1Erecombinant plasmid; (-) control consisted of all cDNA and PCR reagentswithout the addition of cDNA template. Intensity of signal is depictedas the number of plus signs as defined in the figure key.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the 5-HT receptor family is defined as the group ofmammalian proteins that function as receptors for serotonin. A 5-HTreceptor subfamily is defined as a subset of proteins belonging to the5-HT receptor family which are encoded by genes which exhibit homologyof greater than 72% or higher with each other in their deduced aminoacid sequences within presumed transmembrane regions (linearlycontiguous stretches of hydrophobic amino acids, bordered by charged orpolar amino acids, that are long enough to form secondary proteinstructures that span a lipid bilayer). Four human 5-HT receptorsubfamilies can be distinguished based on the information presentlyavailable: 5-HT₁, 5-HT₂, 5-HT₃, and 5-HT₄ (Peroutka, 1991). The 5-HT₂receptor subfamily contains the human 5-HT₂ receptor. Although no otherhuman members of this family have been described, the rat 5-HT₂ receptor(Pritchett, et al. 1988; Julius, et al. Proc. Natl. Acad. Sci. USA87:928-932, 1990) and the rat 5HT_(1C) receptor (Julius, et al. 1988)constitute a rat 5-HT receptor subfamily. The 5-HT₁ subfamily has beensubdivided further as: 5-HT_(1A), 5-HT_(1B), 5-HT_(1C), 5-HT_(1D) (Hamonet al., 1990) and 5-HT_(1E) (Leonhardt et al., 1989). The 5-HT_(1A)subfamily contains the human 5-HT_(1A) receptor, also known as G-21(Fargin, et al. 1988) The 5-HT_(1D) receptor subfamily contains twomembers, the 5-HT_(1D-1) receptor (also termed 5-HT_(1D)α) and the5-HT_(1D-2) receptor (also termed 5-HT_(1D)β). The 5-HT_(1E) subfamilycontains the human 5-HT_(1E) receptor (also termed clone hp75d).Although this definition differs from the pharmacological definitionused earlier, there is significant overlap between the presentdefinition and the pharmacological definition. Members of the 5-HT_(1E)receptor subfamily so described include the 5-HT_(1E) receptor and anyother receptors which have a greater than 72% homology to the DNA andamino acid sequence shown in FIG. 1 according to the definition of"subfamily". This invention relates to the discovery of the first memberof the human 5-HT_(1E) receptor subfamily.

This invention provides an isolated nucleic acid molecule encoding ahuman 5-HT_(1E) receptor. As used herein, the term "isolated nucleicacid molecule" means a nucleic acid molecule that is, a molecule in aform which does not occur in nature. Such a receptor is by definition amember of the 5-HT_(1E) receptor subfamily. Therefore, any receptorwhich meets the defining criteria given above is a human 5-HT_(1E)receptor. One means of isolating a human 5-HT_(1E) receptor is to probea human genomic library with a natural or artificially designed DNAprobe, using methods well known in the art. DNA probes derived from thehuman receptor gene 5-HT_(1E) are particularly useful probes for thispurpose. DNA and cDNA molecules which encode human 5-HT_(1E) receptorsmay be used to obtain complementary genomic DNA, cDNA or RNA from human,mammalian or other animal sources, or to isolate related cDNA or genomicclones by the screening of cDNA or genomic libraries, by methodsdescribed in more detail below. Transcriptional regulatory elements fromthe 5' untranslated region of the isolated clones, and other stability,processing, transcription, translation, and tissuespecificity-determining regions from the 3' and 5' untranslated regionsof the isolated genes are thereby obtained. Examples of a nucleic acidmolecule are an RNA, cDNA, or isolated genomic DNA molecule encoding ahuman 5-HT_(1E) receptor. Such molecules may have coding sequencessubstantially the same as the coding sequence shown in FIG. 1. The DNAmolecule of FIG. 1 encodes the sequence of the human 5-HT_(1E) receptorgene.

This invention further provides a cDNA molecule of encoding a human5-HT_(1E) receptor having a coding sequence substantially the same asthe coding sequence shown in FIG. 1. This molecule is obtained by themeans described above.

This invention also provides an isolated protein which is a human5-HT_(1E) receptor. As used herein, the term "isolated protein means aprotein molecule free of other cellular components. An example of suchprotein is an isolated protein having substantially the same amino acidsequence as the amino acid sequence shown in FIG. 1 which is a human5-HT_(1E) receptor. One means for obtaining isolated 5-HT_(1E) receptoris to express DNA encoding the receptor in a suitable host, such as abacterial, yeast, or mammalian cell, using methods well known in theart, and recovering the receptor protein after it has been expressed insuch a host, again using methods well known in the art. The receptor mayalso be isolated from cells which express it, in particular from cellswhich have been transfected with the expression vectors described belowin more detail.

This invention provides a vector comprising an isolated nucleic acidmolecule such as DNA, RNA, or cDNA encoding a human 5-HT_(1E) receptor.Examples of vectors are viruses such as bacteriophages (such as phagelambda), cosmids, plasmids (such as pUC18, available from Pharmacia,Piscataway, N.J.), and other recombination vectors. Nucleic acidmolecules are inserted into vector genomes by methods well known in theart. For example, insert and vector DNA can both be exposed to arestriction enzyme to create complementary ends on both molecules whichbase pair with each other and are then ligated together with a ligase.Alternatively, linkers can be ligated to the insert DNA which correspondto a restriction site in the vector DNA, which is then digested with therestriction enzyme which cuts at that site. Other means are alsoavailable. A specific example of such plasmids is a plasmid comprisingcDNA having a coding sequence substantially the same as the codingsequence shown in FIG. 1 and designated clone hp75d (Seq. I.D. No. 1).

This invention also provides vectors comprising a DNA molecule encodinga human 5-HT_(1E) receptor, adapted for expression in a bacterial cell,a yeast cell, or a mammalian cell which additionally comprise theregulatory elements necessary for expression of the DNA in thebacterial, yeast, or mammalian cells so located relative to the DNAencoding a human 5-HT_(1E) receptor as to permit expression thereof. DNAhaving coding sequences substantially the same as the coding sequenceshown in FIG. 1 may usefully be inserted into the vectors to expresshuman 5-HT_(1E) receptors. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory,1982). Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors may be obtained commercially orassembled from the sequences described by methods well known in the art,for example the methods described above for constructing vectors ingeneral. Expression vectors are useful to produce cells that express thereceptor. Certain uses for such cells are described in more detailbelow.

This invention further provides a plasmid adapted for expression in abacterial, yeast, or, in particular, a mammalian cell which comprises aDNA molecule encoding a human 5-HT_(1E) receptor and the regulatoryelements necessary for expression of the DNA in the bacterial, yeast, ormammalian cell so located relative to the DNA encoding a human 5-HT_(1E)receptor as to permit expression thereof. Some plasmids adapted forexpression in a mammalian cell are pSVL (available from Pharmacia,Piscataway, N.J.) and pcEXV-3 (Miller J. and Germain R. N., J. Exp. Med.164:1478 (1986)). A specific example of such plasmid is a plasmidadapted for expression in a mammalian cell comprising cDNA having codingsequences substantially the same as the coding sequence shown in FIG. 1and the regulatory elements necessary for expression of the DNA in themammalian cell which is designated pcEXV-hp75d and deposited under ATCCAccession No. 75138. Those skilled in the art will readily appreciatethat numerous plasmids adapted for expression in a mammalian cell whichcomprise DNA of encoding human 5-HT_(1E) receptors and the regulatoryelements necessary to express such DNA in the mammalian cell may beconstructed utilizing existing plasmids and adapted as appropriate tocontain the regulatory elements necessary to express the DNA in themammalian cell. The plasmids may be constructed by the methods describedabove for expression vectors and vectors in general, and by othermethods well known in the art.

The deposit discussed supra, and the other deposits discussed herein,were made pursuant to, and in satisfaction of, the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852. The deposit ofstains bearing accession numbers ATCC 75138, CRL 10913 and CRL 10914were so made Nov. 6, 1991.

This invention provides a mammalian cell comprising a DNA moleculeencoding a human 5-HT_(1E) receptor, such as a mammalian cell comprisinga plasmid adapted for expression in a mammalian cell, which comprises aDNA molecule encoding a human 5-HT_(1E) receptor, the protein encodedthereby is expressed on the cell surface, and the regulatory elementsnecessary for expression of the DNA in the mammalian cell so locatedrelative to the DNA encoding a human 5-HT_(1E) receptor as to permitexpression thereof. Numerous mammalian cells may be used as hosts,including, for example, the mouse fibroblast cell NIH3T3, CHO cells,HeLa cells, Ltk⁻ cells, Y1 cells, etc. A particular example of an Ltk⁻cell is a cell designated 5-HT_(1E) -7 and deposited under ATCCAccession No. CRL 10913 and comprises the plasmid designatedpcEXV-hp75d. Another example is the murine adrenal Y1 cell linedesignated Y-5-HT_(1E) and deposited under ATCC Accession No. CRL 10914.Expression plasmids such as that described supra may be used totransfect mammalian cells by methods well known in the art such ascalcium phosphate precipitation, or DNA encoding these 5-HT_(1E)receptors may be otherwise introduced into mammalian cells, e.g., bymicroinjection, to obtain mammalian cells which comprise DNA, e.g., cDNAor a plasmid, encoding either human 5-HT_(1E) receptor.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a human 5-HT_(1E) receptor can bind toa human 5-HT_(1E) receptor which comprises contacting a mammalian cellcomprising a DNA molecule encoding a human 5-HT_(1E) receptor, theprotein encoded thereby is expressed on the cell surface, with theligand under conditions permitting binding of ligands known to bind tothe 5-HT_(1E) receptor, detecting the presence of any of the ligandbound to the 5-HT_(1E) receptor, and thereby determining whether theligand binds to the 5-HT_(1E) receptor. This invention also provides amethod for determining whether a ligand not known to be capable ofbinding to the human 5-HT_(1E) receptor can functionally activate itsactivity or prevent the action of a ligand which does so. This comprisescontacting a mammalian cell comprising an isolated DNA molecule whichencodes a human 5-HT_(1E) receptor with the ligand under conditionspermitting the activation or blockade of a functional response, detectedby means of a bioassay from the mammalian cell such as a secondmessenger response, and thereby determining whether the ligand activatesor prevents the activation of the human 5-HT_(1E) receptor functionaloutput. The DNA in the cell may have a coding sequence substantially thesame as the coding sequence shown in FIG. 1 preferably, the mammaliancell is nonneuronal in origin. An example of a nonneuronal mammaliancell is an Ltk⁻ cell, in particular the Ltk⁻ cell designated 5-HT_(1E)-7. Another example of a non-neuronal mammalian cell to be used forfunctional assays is a Y1 murine adrenal cell, specifically the Y1 celldesignated Y-5-HT_(1E). The preferred method for determining whether aligand is capable of binding to the human 5-HT_(1E) receptor comprisescontacting a transfected nonneuronal mammalian cell (i.e. a cell thatdoes not naturally express any type of 5-HT or G-protein coupledreceptor, thus will only express such a receptor if it is transfectedinto the cell) expressing a 5-HT_(1E) receptor on its surface, orcontacting a membrane preparation derived from such a transfected cell,with the ligand under conditions which are known to prevail, and thus tobe associated with, in vivo binding of the ligands to a 5-HT_(1E)receptor, detecting the presence of any of the ligand being tested boundto the 5-HT_(1E) receptor on the surface of the cell, and therebydetermining whether the ligand binds to, activates or prevents theactivation of the 5-HT_(1E) receptor. This response system is obtainedby transfection of isolated DNA into a suitable host cell containing thedesired second messenger system such as phosphoinositide hydrolysis,adenylate cyclase, guanylate cyclase or ion channels. Such a host systemis isolated from pre-existing cell lines, or can be generated byinserting appropriate components of second messenger systems intoexisting cell lines. Such a transfection system provides a completeresponse system for investigation or assay of the activity of human5-HT_(1E) receptors with ligands as described above. Transfectionsystems are useful as living cell cultures for competitive bindingassays between known or candidate drugs and ligands which bind to thereceptor and which are labeled by radioactive, spectroscopic or otherreagents. Membrane preparations containing the receptor isolated fromtransfected cells are also useful for these competitive binding assays.Functional assays of second messenger systems or their sequelae intransfection systems act as assays for binding affinity and efficacy inthe activation of receptor function. A transfection system constitutes a"drug discovery system" useful for the identification of natural orsynthetic compounds with potential for drug development that can befurther modified or used directly as therapeutic compounds to activateor inhibit the natural functions of the human 5-HT_(1E) receptor. Thetransfection system is also useful for determining the affinity andefficacy of known drugs at the human 5-HT_(1E) receptor sites.

This invention also provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, the human 5-HT_(1E)receptor on the surface of a cell which comprises contacting a mammaliancell comprising a DNA molecule encoding a human 5-HT_(1E) receptor onthe surface of a cell with a plurality of drugs, determining those drugswhich bind to the mammalian cell, and thereby identifying drugs whichspecifically interact with, and bind to, the human 5-HT_(1E) receptor.This invention also provides a method of screening drugs to identifydrugs which interact with, and activate or block the activation of, thehuman 5-HT_(1E) receptor on the surface of a cell which comprisescontacting the mammalian cell comprising an isolated DNA moleculeencoding and expressing a human 5-HT_(1E) receptor with a plurality ofdrugs, determining those drugs which activate or block the activation ofthe receptor in the mammalian cell using a bioassay such as a secondmessenger assays, and thereby identifying drugs which specificallyinteract with, and activate or block the activation of, a human5-HT.sub. 1E receptor. The DNA in the cell may have a coding sequencesubstantially the same as the coding sequence shown in FIG. 1.Preferably, the mammalian cell is nonneuronal in origin. An example of anonneuronal mammalian cell is an Ltk⁻ cell, in particular the Ltk⁻ celldesignated 5-HT_(1E) -7. Another example of a non-neuronal mammaliancell to be used for functional assays is a Y1 murine adrenal cell,specifically the Y1 cell designated Y-5-HT_(1E). Drug candidates areidentified by choosing chemical compounds which bind with high affinityto the expressed 5-HT_(1E) receptor protein in transfected cells, usingradioligand binding methods well known in the art, examples of which areshown in the binding assays described herein. Drug candidates are alsoscreened for selectivity by identifying compounds which bind with highaffinity to one particular 5-HT_(1E) receptor subtype but do not bindwith high affinity to any other serotonin receptor subtype or to anyother known receptor site. Because selective, high affinity compoundsinteract primarily with the target 5-HT_(1E) receptor site afteradministration to the patient, the chances of producing a drug withunwanted side effects are minimized by this approach. This inventionprovides a pharmaceutical composition comprising a drug identified bythe method described above and a pharmaceutically acceptable carrier. Asused herein, the term "pharmaceutically acceptable carrier" encompassesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, and emulsions, such as an oil/water orwater/oil emulsion, and various types of wetting agents. Once thecandidate drug has been shown to be adequately bio-available following aparticular route of administration, for example orally or by injection(adequate therapeutic concentrations must be maintained at the site ofaction for an adequate period to gain the desired therapeutic benefit),and has been shown to be non-toxic and therapeutically effective inappropriate disease models, the drug may be administered to patients bythat route of administration determined to make the drug bio-available,in an appropriate solid or solution formulation, to gain the desiredtherapeutic benefit.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1E) receptor, for example with a coding sequenceincluded within the sequence shown in FIG. 1. As used herein, the phrase"specifically hybridizing" means the ability of a nucleic acid moleculeto recognize a nucleic acid sequence complementary to its own and toform double-helical segments through hydrogen bonding betweencomplementary base pairs. Nucleic acid probe technology is well known tothose skilled in the art who will readily appreciate that such probesmay vary greatly in length and may be labeled with a detectable label,such as a radioisotope or fluorescent dye, to facilitate detection ofthe probe. Detection of nucleic acid encoding human 5-HT_(1E) receptorsis useful as a diagnostic test for any disease process in which levelsof expression of the corresponding 5-HT_(1E) receptor is altered. DNAprobe molecules are produced by insertion of a DNA molecule whichencodes human 5-HT_(1E) receptor or fragments thereof into suitablevectors, such as plasmids or bacteriophages, followed by insertion intosuitable bacterial host cells and replication and harvesting of the DNAprobes, all using methods well known in the art. For example, the DNAmay be extracted from a cell lysate using phenol and ethanol, digestedwith restriction enzymes corresponding to the insertion sites of the DNAinto the vector (discussed above), electrophoresed, and cut out of theresulting gel. An example of such DNA molecule is shown in FIG. 1. Theprobes are useful for `in situ` hybridization or in order to locatetissues which express this gene family, or for other hybridizationassays for the presence of these genes or their mRNA in variousbiological tissues. In addition, synthesized oligonucleotides (producedby a DNA synthesizer) complementary to the sequence of a DNA moleculewhich encodes human 5-HT_(1E) receptor of are useful as probes for thesegenes, for their associated mRNA, or for the isolation of related genesby homology screening of genomic or cDNA libraries, or by the use ofamplification techniques such as the Polymerase Chain Reaction.Synthesized oligonucleotides as described may also be used to determinethe cellular localization of the mRNA produced by the 5-HT_(1E) gene byin situ hybridization. An example of such an oligonucleotide is:GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT(Seq. I.D. No. 8).

This invention also provides a method of detecting expression of a5-HT_(1E) receptor on the surface of a cell by detecting the presence ofmRNA coding for a 5-HT_(1E) receptor which comprises obtaining totalmRNA from the cell using methods well known in the art and contactingthe mRNA so obtained with a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1E) receptor under hybridizing conditions,detecting the presence of mRNA hybridized to the probe, and therebydetecting the expression of the 5-HT_(1E) receptor by the cell.Hybridization of probes to target nucleic acid molecules such as mRNAmolecules employs techniques well known in the art. In one possiblemeans of performing this method, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing a column which binds the poly-A tails of the mRNA molecules. ThemRNA is then exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a human 5-HT_(1E) receptor so as to prevent translation ofthe mRNA molecule. The antisense oligonucleotide may have a sequencecapable of binding specifically with any sequences of the cDNA moleculewhose sequence is shown in FIG. 1. As used herein, the phrase "bindingspecifically" means the ability of a nucleic acid sequence to recognizea nucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs. A particular example of an antisense oligonucleotide is anantisense oligonucleotide comprising chemical analogues of nucleotides.

This invention also provides a pharmaceutical composition comprising anamount of the oligonucleotide described above effective to reduceexpression of a human 5-HT_(1E) receptor by passing through a cellmembrane and binding specifically with mRNA encoding a human 5-HT_(1E)receptor in the cell so as to prevent its translation and apharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane. The oligonucleotide may be coupled to asubstance which inactivates mRNA, such as a ribozyme. Thepharmaceutically acceptable hydrophobic carrier capable of passingthrough cell membranes may also comprise a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type. The structure may be part of a proteinknown to bind a cell-type specific receptor, for example an insulinmolecule, which would target pancreatic cells. DNA molecules havingcoding sequences substantially the same as the coding sequence shown inFIG. 1 may be used as the oligonucleotides of the pharmaceuticalcomposition.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of expression of a 5-HT_(1E) receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to reduce expression of the5-HT_(1E) receptor by the subject. This invention further provides amethod of treating an abnormal condition related to 5-HT_(1E) receptoractivity which comprises administering to a subject an amount of thepharmaceutical composition described above effective to reduceexpression of the 5-HT_(1E) receptor by the subject. Several examples ofsuch abnormal conditions are dementia, Parkinson's disease, feedingdisorders, pathological anxiety, schizophrenia, or a migraine headache.

Antisense oligonucleotide drugs inhibit translation of mRNA encodingthese receptors. Synthetic oligonucleotides, or other antisense chemicalstructures are designed to bind to mRNA encoding the 5-HT_(1E) receptorand inhibit translation of mRNA and are useful as drugs to inhibitexpression of 5-HT_(1E) receptor genes in patients. This inventionprovides a means to therapeutically alter levels of expression of human5-HT_(1E) receptors by the use of a synthetic antisense oligonucleotidedrug (SAOD) which inhibits translation of mRNA encoding these receptors.Synthetic oligonucleotides, or other antisense chemical structuresdesigned to recognize and selectively bind to mRNA, are constructed tobe complementary to portions of the nucleotide sequences shown in FIG. 1of DNA, RNA or of chemically modified, artificial nucleic acids. TheSAOD is designed to be stable in the blood stream for administration topatients by injection, or in laboratory cell culture conditions, foradministration to cells removed from the patient. The SAOD is designedto be capable of passing through cell membranes in order to enter thecytoplasm of the cell by virtue of physical and chemical properties ofthe SAOD which render it capable of passing through cell membranes (e.g.by designing small, hydrophobic SAOD chemical structures) or by virtueof specific transport systems in the cell which recognize and transportthe SAOD into the cell. In addition, the SAOD can be designed foradministration only to certain selected cell populations by targetingthe SAOD to be recognized by specific cellular uptake mechanisms whichbinds and takes up the SAOD only within certain selected cellpopulations. For example, the SAOD may be designed to bind to a receptorfound only in a certain cell type, as discussed above. The SAOD is alsodesigned to recognize and selectively bind to the target mRNA sequence,which may correspond to a sequence contained within the sequence shownin FIG. 1 by virtue of complementary base pairing to the mRNA. Finally,the SAOD is designed to inactivate the target mRNA sequence by any ofthree mechanisms: 1) by binding to the target mRNA and thus inducingdegradation of the mRNA by intrinsic cellular mechanisms such as RNAse Idigestion, 2) by inhibiting translation of the mRNA target byinterfering with the binding of translation-regulating factors or ofribosomes, or 3) by inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups, which either degrade orchemically modify the target mRNA. Synthetic antisense oligonucleotidedrugs have been shown to be capable of the properties described abovewhen directed against mRNA targets (J. S. Cohen, Trends in Pharm. Sci.10, 435 (1989); H. M. Weintraub, Sci. Am. January (1990) p. 40). Inaddition, coupling of ribozymes to antisense oligonucleotides is apromising strategy for inactivating target mRNA (N. Sarver et al.,Science 247, 1222 (1990)). An SAOD serves as an effective therapeuticagent if it is designed to be administered to a patient by injection, orif the patient's target cells are removed, treated with the SAOD in thelaboratory, and replaced in the patient. In this manner, an SAOD servesas a therapy to reduce receptor expression in particular target cells ofa patient, in any clinical condition which may benefit from reducedexpression of 5-HT_(1E) receptors.

This invention provides an antibody directed to the human 5-HT_(1E)receptor, for example a monoclonal antibody directed to an epitope of ahuman 5-HT_(1E) receptor present on the surface of a cell and having anamino acid sequence substantially the same as an amino acid sequence fora cell surface epitope of the human 5-HT_(1E) receptor included in theamino acid sequence shown in FIG. 1 (Seq. I.D. No. 2). Amino acidsequences may be analyzed by methods well known in the art to determinewhether they produce hydrophobic or hydrophilic regions in the proteinswhich they build. In the case of cell membrane proteins, hydrophobicregions are well known to form the part of the protein that is insertedinto the lipid bilayer which forms the cell membrane, while hydrophilicregions are located on the cell surface, in an aqueous environment.Therefore antibodies to the hydrophilic amino acid sequences shown inFIG. 1 will bind to a surface epitope of a human 5-HT_(1E) receptor, asdescribed. Antibodies directed to human 5-HT_(1E) receptors may beserum-derived or monoclonal and are prepared using methods well known inthe art. For example, monoclonal antibodies are prepared using hybridomatechnology by fusing antibody producing B cells from immunized animalswith myeloma cells and selecting the resulting hybridoma cell lineproducing the desired antibody. Cells such as NIH3T3 cells or Ltk⁻ cellsmay be used as immunogens to raise such an antibody. Alternatively,synthetic peptides may be prepared using commercially available machinesand the amino acid sequence shown in FIG. 1 (Seq. I.D. No. 2). As astill further alternative, DNA, such as a cDNA or a fragment thereof,may be cloned and expressed and the resulting polypeptide recovered andused as an immunogen. These antibodies are useful to detect the presenceof human 5-HT_(1E) receptors encoded by the isolated DNA, or to inhibitthe function of the receptors in living animals, in humans, or inbiological tissues or fluids isolated from animals or humans.

This invention provides a pharmaceutical composition which comprises anamount of an antibody directed to the human 5-HT_(1E) receptor effectiveto block binding of naturally occurring ligands to the 5-HT_(1E)receptor, and a pharmaceutically acceptable carrier. A monoclonalantibody directed to an epitope of a human 5-HT_(1E) receptor present onthe surface of a cell and having an amino acid sequence substantiallythe same as an amino acid sequence for a cell surface epitope of thehuman 5-HT_(1E) receptor included in the amino acid sequence shown inFIG. 1 is useful for this purpose.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of expression of a human 5-HT_(1E) receptorwhich comprises administering to a subject an amount of thepharmaceutical composition described above effective to block binding ofnaturally occurring ligands to the 5-HT_(1E) receptor and therebyalleviate abnormalities resulting from overexpression of a human5-HT_(1E) receptor. Binding of the antibody to the receptor prevents thereceptor from functioning, thereby neutralizing the effects ofoverexpression. The monoclonal antibodies described above are bothuseful for this purpose. This invention additionally provides a methodof treating an abnormal condition related to an excess of 5-HT_(1E)receptor activity which comprises administering to a subject an amountof the pharmaceutical composition described above effective to blockbinding of naturally occurring ligands to the 5-HT_(1E) receptor andthereby alleviate the abnormal condition. Some examples of abnormalconditions are dementia, Parkinson's disease, feeding disorders,pathological anxiety, schizophrenia, and a migraine headache.

This invention provides a method of detecting the presence of a5-HT_(1E) receptor on the surface of a cell which comprises contactingthe cell with an antibody directed to the human 5-HT_(1E) receptor,under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebythe presence of the human 5-HT_(1E) receptor on the surface of the cell.Such a method is useful for determining whether a given cell isdefective in expression of 5-HT_(1E) receptors on the surface of thecell. Bound antibodies are detected by methods well known in the art,for example by binding fluorescent markers to the antibodies andexamining the cell sample under a fluorescence microscope to detectfluorescence on a cell indicative of antibody binding. The monoclonalantibodies described above are useful for this purpose. This inventionprovides a transgenic nonhuman mammal expressing DNA encoding a human5-HT_(1E) receptor. This invention also provides a transgenic nonhumanmammal expressing DNA encoding a human 5-HT_(1E) receptor so mutated asto be incapable of normal receptor activity, and not expressing native5-HT_(1E) receptor. This invention also provides a transgenic nonhumanmammal whose genome comprises antisense DNA complementary to DNAencoding a human 5-HT_(1E) receptor so placed as to be transcribed intoantisense mRNA which is complementary to mRNA encoding a 5-HT_(1E)receptor and which hybridizes to mRNA encoding a 5-HT_(1E) receptorthereby reducing its translation. The DNA may additionally comprise aninducible promoter or additionally comprise tissue specific regulatoryelements, so that expression can be induced, or restricted to specificcell types. Examples of DNA are DNA or cDNA molecules having a codingsequence substantially the same as the coding sequence shown in FIG. 1.An example of a transgenic animal is a transgenic mouse. Examples oftissue specificity-determining regions are the metallothionein promotor(Low, M. J., Lechan, R. M., Hammer, R. E. et al. Science 231:1002-1004(1986)) and the L7 promotor (Oberdick, J., Smeyne, R. J., Mann, J. R.,Jackson, S. and Morgan, J. I. Science 248:223-226 (1990)).

Animal model systems which elucidate the physiological and behavioralroles of human 5-HT_(1E) receptors are produced by creating transgenicanimals in which the expression of a 5-HT_(1E) receptor is eitherincreased or decreased, or the amino acid sequence of the expressed5-HT_(1E) receptor protein is altered, by a variety of techniques.Examples of these techniques include: 1) Insertion of normal or mutantversions of DNA encoding a human 5-HT_(1E) receptor or homologous animalversions of these genes, by microinjection, retroviral infection orother means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal (Hogan B. etal. Manipulating the Mouse Embryo, A Laboratory Manual, Cold SpringHarbor Laboratory (1986)). 2) Homologous recombination (Capecchi M. R.Science 244:1288-1292 (1989); Zimmer, A. and Gruss, P. Nature338:150-153 (1989)) of mutant or normal, human or animal versions ofthese genes with the native gene locus in transgenic animals to alterthe regulation of expression or the structure of these 5-HT_(1E)receptors. The technique of homologous recombination is well known inthe art. It replaces the native gene with the inserted gene and so isuseful for producing an animal that cannot express native receptor butdoes express, for example, an inserted mutant receptor, which hasreplaced the native receptor in the animal's genome by recombination,resulting in underexpression of the receptor. Microinjection adds genesto the genome, but does not remove them, and so is useful for producingan animal which expresses its own and added receptors, resulting inoverexpression of the receptor. One means available for producing atransgenic animal, with a mouse as an example, is as follows: Femalemice are mated, and the resulting fertilized eggs are dissected out oftheir oviducts. The eggs are stored in an appropriate medium such as M2medium (Hogan B. et al. Manipulating the Mouse Embryo, A LaboratoryManual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding ahuman 5-HT_(1E) receptor is purified from a vector (such as plasmidpCEXV-hp75d described above) by methods well known in the art. Induciblepromoters may be fused with the coding region of the DNA to provide anexperimental means to regulate expression of the trans-gene.Alternatively or in addition, tissue specific regulatory elements may befused with the coding region to permit tissue-specific expression of thetrans-gene. The DNA, in an appropriately buffered solution, is put intoa microinjection needle (which may be made from capillary tubing using apipet puller) and the egg to be injected is put in a depression slide.The needle is inserted into the pronucleus of the egg, and the DNAsolution is injected. The injected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

Since the normal action of receptor-specific drugs is to activate or toinhibit the receptor, the transgenic animal model systems describedabove are useful for testing the biological activity of drugs directedagainst these 5-HT_(1E) receptors even before such drugs becomeavailable. These animal model systems are useful for predicting orevaluating possible therapeutic applications of drugs which activate orinhibit these 5-HT_(1E) receptors by inducing or inhibiting expressionof the native or trans-gene and thus increasing or decreasing expressionof normal or mutant 5-HT_(1E) receptors in the living animal. Thus, amodel system is produced in which the biological activity of drugsdirected against these 5-HT_(1E) receptors are evaluated before suchdrugs become available. The transgenic animals which over or underproduce the 5-HT_(1E) receptor indicate by their physiological statewhether over or under production of the 5-HT_(1E) receptor istherapeutically useful. It is therefore useful to evaluate drug actionbased on the transgenic model system. One use is based on the fact thatit is well known in the art that a drug such as an antidepressant actsby blocking neurotransmitter uptake, and thereby increases the amount ofneurotransmitter in the synaptic cleft. The physiological result of thisaction is to stimulate the production of less receptor by the affectedcells, leading eventually to underexpression. Therefore, an animal whichunderexpresses receptor is useful as a test system to investigatewhether the actions of such drugs which result in under expression arein fact therapeutic. Another use is that if overexpression is found tolead to abnormalities, then a drug which down-regulates or acts as anantagonist to 5-HT_(1E) receptor is indicated as worth developing, andif a promising therapeutic application is uncovered by these animalmodel systems, activation or inhibition of the 5-HT_(1E) receptor isachieved therapeutically either by producing agonist or antagonist drugsdirected against these 5-HT_(1E) receptors or by any method whichincreases or decreases the expression of these 5-HT_(1E) receptors inman.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1E) receptors whichcomprises producing a transgenic nonhuman animal whose levels of human5-HT_(1E) receptor expression are varied by use of an inducible promoterwhich regulates human 5-HT_(1E) receptor expression. This invention alsoprovides a method of determining the physiological effects of expressingvarying levels of human 5-HT_(1E) receptors which comprises producing apanel of transgenic nonhuman animals each expressing a different amountof human 5-HT_(1E) receptor. Such animals may be produced by introducingdifferent amounts of DNA encoding a human 5-HT_(1E) receptor into theoocytes from which the transgenic animals are developed.

This invention also provides a method for identifying a substancecapable of alleviating abnormalities resulting from overexpression of ahuman 5-HT_(1E) receptor comprising administering the substance to atransgenic nonhuman mammal expressing at least one artificiallyintroduced DNA molecule encoding a human 5-HT_(1E) receptor anddetermining whether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofoverexpression of a human 5-HT_(1E) receptor. As used herein, the term"substance" means a compound or composition which may be natural,synthetic, or a product derived from screening. Examples of DNAmolecules are DNA or cDNA molecules having a coding sequencesubstantially the same as the coding sequence shown in FIG. 1.

This invention provides a pharmaceutical composition comprising anamount of the substance described supra effective to alleviate theabnormalities resulting from overexpression of 5-HT_(1E) receptor and apharmaceutically acceptable carrier.

This invention further provides a method for treating the abnormalitiesresulting from overexpression of a human 5-HT_(1E) receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to alleviate the abnormalitiesresulting from overexpression of a human 5-HT_(1E) receptor.

This invention provides a method for identifying a substance capable ofalleviating the abnormalities resulting from underexpression of a human5-HT_(1E) receptor comprising administering the substance to thetransgenic nonhuman mammal described above which expresses onlynonfunctional human 5-HT_(1E) receptor and determining whether thesubstance alleviates the physical and behavioral abnormalities displayedby the transgenic nonhuman mammal as a result of underexpression of ahuman 5-HT_(1E) receptor.

This invention also provides a pharmaceutical composition comprising anamount of a substance effective to alleviate abnormalities resultingfrom underexpression of 5-HT_(1E) receptor and a pharmaceuticallyacceptable carrier.

This invention further provides a method for treating the abnormalitiesresulting from underexpression of a human 5-HT_(1E) receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to alleviate the abnormalitiesresulting from underexpression of a human 5-HT_(1E) receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the expression of a specific human 5-HT_(1E)receptor allele which comprises: a) obtaining DNA of subjects sufferingfrom the disorder: b) performing a restriction digest of the DNA with apanel of restriction enzymes; c.electrophoretically separating theresulting DNA fragments on a sizing gel: d) contacting the resulting gelwith a nucleic acid probe capable of specifically hybridizing to DNAencoding a human 5-HT_(1E) receptor and labelled with a detectablemarker; e) detecting labelled bands which have hybridized to the DNAencoding a human 5-HT_(1E) receptor label led with a detectable markerto create a unique band pattern specific to the DNA of subjectssuffering from the disorder; f) preparing DNA obtained for diagnosis bysteps a-e; and g) comparing the unique band pattern specific to the DNAof subjects suffering from the disorder from step e and the DNA obtainedfor diagnosis from step f to determine whether the patterns are the sameor different and thereby to diagnose predisposition to the disorder ifthe patterns are the same. This method may also be used to diagnose adisorder associated with the expression of a specific human 5-HT_(1E)receptor allele.

This invention provides a method of preparing the isolated 5-HT_(1E)receptor which comprises inducing cells to express 5-HT_(1E) receptor,recovering the receptor from the resulting cells, and purifying thereceptor so recovered. An example of an isolated 5-HT_(1E) receptor isan isolated protein having substantially the same amino acid sequence asthe amino acid sequence shown in FIG. 1. For example, cells can beinduced to express receptors by exposure to substances such as hormones.The cells can then be homogenized and the receptor isolated from thehomogenate using an affinity column comprising, for example, serotoninor another substance which is known to bind to the receptor. Theresulting fractions can then be purified by contacting them with an ionexchange column, and determining which fraction contains receptoractivity or binds anti-receptor antibodies.

This invention provides a method of preparing the isolated 5-HT_(1E)receptor which comprises inserting nucleic acid encoding 5-HT_(1E)receptor in a suitable vector, inserting the resulting vector in asuitable host cell, recovering the receptor produced by the resultingcell, and purifying the receptor so recovered. An example of an isolated5-HT_(1E) receptor is an isolated protein having substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 1. Thismethod for preparing 5-HT_(1E) receptor uses recombinant DNA technologymethods well known in the art. For example, isolated nucleic acidencoding 5-HT_(1E) receptor is inserted in a suitable vector, such as anexpression vector. A suitable host cell, such as a bacterial cell, or aeukaryotic cell such as a yeast cell, is transfected with the vector.5-HT_(1E) receptor is isolated from the culture medium by affinitypurification or by chromatography or by other methods well known in theart.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a receptor so as to prevent translation of the mRNAmolecule.

This invention also provides a transgenic nonhuman mammal expressing DNAencoding a receptor.

This invention further provides a transgenic nonhuman mammal expressingDNA encoding a receptor so mutated as to be incapable of normal receptoractivity, and not expressing native receptor.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a transgenic nonhuman animal whose levels of receptorexpression are varied by use of an inducible promoter which regulatesreceptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a panel of transgenic nonhuman animals each expressing adifferent amount of the receptor.

This invention further provides transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a receptor soplaced as to be transcribed into antisense mRNA which is complementaryto mRNA encoding the receptor and which hybridizes to mRNA encoding thereceptor thereby preventing its translation.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a receptor can bind to a receptorwhich comprises contacting a mammalian cell comprising an isolated DNAmolecule encoding the receptor with the ligand under conditionspermitting binding of ligands known to bind to a receptor, detecting thepresence of any of the ligand bound to the receptor, and therebydetermining whether the ligand binds to the receptor.

Applicants have identified individual receptor subtype proteins and havedescribed methods for the identification of pharmacological compoundsfor therapeutic treatments. Pharmacological compounds which are directedagainst specific receptor subtypes provide effective new therapies withminimal side effects.

This invention identifies for the first time a new receptor protein, itsamino acid sequence, and its human gene. Furthermore, this inventiondescribes a previously unrecognized group of receptors within thedefinition of a 5-HT_(1E) receptor. The information and experimentaltools provided by this discovery are useful to generate new therapeuticagents, and new therapeutic or diagnostic assays for this new receptorprotein, its associated mRNA molecule or its associated genomic DNA. Theinformation and experimental tools provided by this discovery will beuseful to generate new therapeutic agents, and new therapeutic ordiagnostic assays for this new receptor protein, its associated mRNAmolecule, or its associated genomic DNA.

Specifically, this invention relates to the first isolation of a humancDNA and genomic clone encoding a 5-HT_(1E) receptor. A new human genefor the receptor identified herein as 5-HT_(1E) has been identified andcharacterized, and a series of related cDNA and genomic clones have beenisolated. In addition, the human 5-HT_(1E) receptor has been expressedin Ltk⁻ cells and Y1 cells by transfecting the cells with the plasmidpcEXV-hp75d. The pharmacological binding properties of the proteinencoded have been determined, and these binding properties classify thisprotein as a serotonin 5-HT_(1E) receptor. Mammalian cell linesexpressing this human 5-HT_(1E) receptor at the cell surface have beenconstructed, thus establishing the first well-defined, cultured celllines with which to study this 5-HT_(1E) receptor.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrativeof the invention as described more fully in the claims which followthereafter.

MATERIALS and METHODS

Cloning and Sequencing

A human placental genomic library (Stratagene) was screened usingoligonucleotides derived from the human 5-HT_(1D)β receptor gene (U.S.Ser. No. 520,716) , as a probe. Overlapping oligomers complementary tothe 5-HT_(1D)β sequence in transmembrane domains III, V and VI werelabeled with ³² P-dATP and ³² P-dCTP by synthesis with the largefragment of DNA Polymerase (Maniatis et al., 1982). Hybridization wasperformed at 40° C. in a solution containing 25% formamide, 10% dextransulfate, 5× SSC (1× SSC is 0.15M sodium chloride, 0.015M sodiumcitrate), 1× Denhardt's (0.02% polyvinylpyrrolidone, 0.02% Ficoll, and0.02% bovine serum albumin), and 200 μg/ml of sonicated salmon spermDNA. The filters were washed at 40° C. in 0.1× SSC containing 0.1%sodium dodecyl sulfate (SDS) and exposed at -70° C. to Kodak XAR film inthe presence of an intensifying screen. Lambda phage hybridizing to theprobe were plaque purified and DNA was prepared for Southern blotanalysis (Southern, 1975; Maniatis et al., 1982). For subcloning andfurther Southern blot analysis DNA was inserted into pUC18 (Pharmacia,Piscataway, N.J.). Nucleotide sequence analysis was done by the Sangerdideoxy nucleotide chain-termination method (Sanger 1977) on denatureddouble-stranded plasmid templates using Sequenase (U.S. BiochemicalCorp., Cleveland, Ohio) .

Expression

The entire coding region of clone hp75d was cloned into the eukaryoticexpression vector pcEXV-3 (Miller, 1986). Stable cell lines wereobtained by cotransfection with the plasmid pcEXV-3 (containing the5-HT_(1E) receptor gene) and the plasmid pGCcos3neo (containing theaminoglycoside transferase gene) into Ltk⁻ cells and Y1 cells usingcalcium phosphate (reagents obtained from Specialty Media, Lavellette,N.J.). The cells were grown in a controlled environment (37° C., 5% CO₂)as monolayers in Dulbecco's modified Eagle medium (Gibco, Grand Island,N.Y.) or Flo Medium Specialty Media, Inc., Lavallette, N.J.) containing25 mM glucose and supplemented with 10% bovine calf serum, 100 U/mlpenicillin G and 100 μg/ml streptomycin sulfate. Stable clones were thenselected for resistance to the antibiotic G-418 and harvested membraneswere screened for their ability to bind [³ H] serotonin.

Detection of 5-HT_(1E) Receptor mRNA in Brain Using PCR

Total RNA from human brain tissue was extracted using the RNasolprotocol as described by the manufacturer. cDNA was prepared from 5 μgof total RNA with random hexanucleotide primers (500 pmoles) usingSuperscript reverse transcriptase (BRL, MD.) in 50 mM Tris-HCL pH 8.3buffer containing 40 u RNasin, 2.5 mM MgCl₂, 50 mM KCL and 1 mM dNTPs,at 42° C. for 1 hr. The reaction was stopped by heating at 95° C. for 5min. and chilled on ice. After terminating the reaction, RNase H (2 u)was added and incubated at 37° C. for 20 min. An aliquot (1 μg) of thefirst strand cDNA was diluted (1:5) into a 50 μl PCR reaction mixture(200 μM dNTPs final concentration) containing 1.25 U of Taq polymerase(Cetus Corp., Calif.) in the buffer supplied by the manufacturer, and 1μM of 5' primer= 5' TACCACGCGGCCAAGAGCCTTTACCA 3' (Seq. I.D. No. 9) and3' primer=5'TGGTGCTAGAGATCTGCTGACGTTC 3' (Seq. I.D. No. 10),oligonucleotides derived from the third cytoplasmic loop region. The PCRamplification reaction was carried out on a Perkin Elmer Cetus thermalcycler by first a 5 min. incubation at 95° C. followed by 30 rounds ofthe following cycle: 2 min. at 94° C., 2 min. at 42° C., 3 min. at 72°C., with a 3" extension, followed at the end by a 15 min. incubation at72° C. In order to control for the amplification of DNA (carried overduring the RNA extraction), control PCR reactions were run in parallelwith RNA diluted in the same manner as the cDNA samples. If necessary,RNA samples were pretreated with RNase-free DNase to eliminate anycontaminating DNA. Positive controls were included in all experiments,consisting of plasmid containing 5HT1E receptor gene sequences. Theproducts of the PCR amplification were separated by electrophoresis in1.5% agarose. Amplified fragments were identified by blotting the gel tonitrocellulose and probing with an oligonucleotide internal to the 5'-and 3'-primers (5'GAGAAGTCAGACACACAGAAAGTCTGTGTAAGTTTTACAACTTGC 3')(Seq. I.D. No. 11) and end-labeled with [c-³² P]ATP using T₄polynucleotide kinase. Hybridization was performed at 40° C. in asolution containing 50% formamide, 10% dextran sulfate, 5× SSC (1 × SSCis 0.15M sodium chloride, 0.015M sodium citrate), 1× Denhardt's (0.02%polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and200 μg/ml of sonicated salmon sperm DNA. The filters were washed at 50°C. in 0.1× SSC containing 0.1% sodium dodecyl sulfate and exposed at 70°C. to Kodak XAR film in the presence of an intensifying screen.

Membrane Preparation

Membranes were prepared from transfected Ltk⁻ cells which were grown to100% confluency. The cells were washed twice with phosphate-bufferedsaline, scraped from the culture dishes into 5 ml of ice-coldphosphate-buffered saline, and centrifuged at 200×g for 5 min at 4° C.The pellet was resuspended in 2.5 ml of ice-cold Tris buffer (20 mM Tris-HCl, pH 7.4 at 23° C., 5 mM EDTA) and homogenized by a Wheaton tissuegrinder. The lysate was subsequently centrifuged at 200×g for 5 min at4° C. to pellet large fragments which were discarded. The supernatantwas collected and centrifuged at 40,000× g for 20 min at 4° C. Thepellet resulting from this centrifugation was washed once in ice-coldTris buffer and finally resuspended in a final buffer containing 50 mMTris-HCl and 0.5 mM EDTA, pH 7.4 at 23° C. Membrane preparations werekept on ice and utilized within two hours for the radioligand bindingassays. Protein concentrations were determined by the method of Bradford(1976) using bovine serum albumin as the standard.

Radioligand Binding [³ H]5HT binding was performed using slightmodifications of the 5-HT_(1E) assay conditions reported by Leonhardt etal. (1989) with the omission of masking ligands. Radioligand bindingstudies were achieved at 37° C. in a total volume of 250 μl of buffer(50 mM Tris, 10 mM MgCl₂, 0.2 mM EDTA, 10 μM pargyline, 0.1% ascorbate,pH 7.4 at 37° C.) in 96 well microtiter plates. Saturation studies wereconducted using [³ H]5-HT at 12 different concentrations ranging from0.5 nM to 100 nM. Displacement studies were performed using 4.5-5.5 nM[³ H]5-HT. The binding profile of drugs in competition experiments wasaccomplished using 10-12 concentrations of compound. Incubation timeswere 30 min for both saturation and displacement studies based uponinitial investigations which determined equilibrium binding conditions.Nonspecific binding was defined in the presence of 10 μM 5-HT. Bindingwas initiated by the addition of 50 μl membrane homogenates (10-20 μg).The reaction was terminated by rapid filtration through presoaked (0.5%polyethyleneimine) filters using 48R Cell Brandel Harvester(Gaithersburg, Md.). Subsequently, filters were washed for 5 secondswith ice cold buffer (50 mM Tris HCL, pH 7.4 at 4° C.), dried and placedinto vials containing 2.5 ml of Readi-Safe (Beckman, Fullerton, Calif.)and radioactivity was measured using a Beckman LS 5000TA liquidscintillation counter. The efficiency of counting of [³ H]5HT averagedbetween 45-50%. Binding data was analyzed by computer-assisted nonlinearregression analysis (Accufit and Accucomp, Lundon Software, ChagrinFalls, Ohio). IC₅₀ values were converted to Ki values using theCheng-Prusoff equation (1973). All experiments were performed intriplicate.

Measurements of cAMP Formation

Transfected Y1 cells (expression level=688 fmol/mg of protein) wereincubated in DMEM, 5 mM theophylline, 10 mM Hepes(4-[2-Hydroxyethyl]-1-piperazineethanesulfonic acid), 10 μM pargyline,for 20 minutes at 37° C., 5% CO₂. A 5-HT dose-effect curve was thenconducted by adding 12 different final concentrations of drug rangingfrom 10 uM to 0.1 nM, followed immediately by the addition of forskolin(10 μM). Subsequently, the cells were incubated for an additional 10minutes at 37° C., 5% CO₂. The media was aspirated and the reactionterminated by the addition of 100 mM HCl. The plates were stored at 4°C. for 15 minutes and centrifuged for 5 minutes (500×g at 4° C.) topellet cellular debris. Aliquots of the supernatant fraction were thenstored at -20° C. prior to assessment of cAMP formation byradioimmunoassay (cAMP Radioimmunoassay kit, Advanced Magnetics,Cambridge, Mass.).

Detection of 5-HT_(1E) Receptor mRNA in Brain Using in SituHybridization Histochemistry

Labeling of 5-HT_(1E) receptor oligo probe with digoxigenin-11-dUTP: The5-HT_(1E) receptor oligo probe (45 mer sequence:GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT) (Seq. I.D. No. 12) wassynthesized in Molecular Biology Department of Neurogenetic Corp. The 3'end tailing reaction was as follows: To a sterile 1.5 ml Eppendorf tubeadd (1) 8 μl of 5-HT_(1E) receptor oligo probe (520 ng, denatured at 95°C., then chilled on ice); (2) 8 μl of 5× tailing buffer (BRL); (3) 5 μlof digoxigenin-11-dUTP (Boehringer); (4) 4 μl of dATP at 1/50 dilution;(5) 7 μl of terminal transferase (TdT, BRL); (6) 7 μl of distilledwater. The above reaction mixture was incubated at 37° C. for 5 min. Thetailed oligo probe was purified by ethanol precipitation, vacuum driedand reconstituted in 40 μl of distilled water.

Tissue Preparation

Guinea pig brains were dissected and frozen, first in methylbutane, thenon dry ice. Brain sections were cut at 11 μm on a cryostat, thaw-mountedon gelatin-coated slides, and stored at -80° C. On the day of theexperiment, the brain sections were quickly brought to room temperatureusing a cool air stream, and fixed in 3% paraformaldehyde made up in0.1M PBS containing 0.02% diethylpyrocarbonate (DEPC). After rinsed in0.1M PBS and 2× SSC, the tissue sections were dehydrated in gradedethanol and air dried.

Prehybridization

All the tissue sections were pre-incubated with hybridization buffer ina humid chamber at 25° C. for 1 hr. Twenty ml of the hybridizationbuffer contained (1). 10 ml of 100% formamide; (2) 4 ml of 20×SSC; (3)0.4 ml of 50× Denhardts; (4) 1.0 ml of 10 mg/ml salmon sperm DNA; (5)0.5 ml of 10 mg/ml yeast tRNA; (6) 4 ml of Dextran sulfate.

Hybridization

The digoxigenin labeled 5-HT_(1E) oligo probe (40 μl) was diluted in 1ml of hybridization buffer. Each tissue section was then covered with0.1 ml of the hybridization buffer with probe and incubated at 40° C.for 82 hr. After incubation all the sections were washed in 2× SSC (25°C., 1 hr), in 1× SSC (25° C., 1 hr), finally in 0.5× SSC, first 37° C.30 min, then 25° C. 30 min.

Immunological Detection

Following the post-hybridization washes slides were rinsed in Buffer #1(100 mM Tris-HCl; 150 mM NaCl; pH 7.5) for 1 min, then incubated with 2%normal sheep serum plus 0.3% triton X-100 in Buffer #1 for 30 min. Afterthis preparation the sections were incubated with anti-digoxigeninconjugated with alkaline phosphatase at dilution 1:500 in Buffer #1containing 1% normal sheep serum and 0.3% triton X-100 at RT for 1.5 hr,then at 4° C. overnight. The next day the tissue sections were rinsed inBuffer #1 for 10 min, then in Buffer #2 (100 mM Tris-HCl; 100 mM NaCl;50 mM MgCl₂ ; pH 9.5) for 10 min. The chromagen solution was preparedimmediately before the colorimetric reaction: i.e. to 10 ml of Buffer#2, (1) 45 μl of 4 -nitro blue tetrazolium chloride (NBT, 75 mg/ml in70% dimethylformamide); (2) 35 μl of5-bromo-4-chloro-3-indolyl-phosphate (x-phosphate, 50 mg/ml in 100%dimethylformamide); and (3) 2.5 mg of levamisole were added. In order tocarry out the colorimetric reaction sections were incubated with thechromogen solution in a humid, light-tight box overnight. The chromogenreaction was halted by rinsing the slides in Buffer #3 (10 mM Tris-HCl;1 mM EDTA; pH 8.0). The sections were then rinsed in PBS, covered withAquamount, and examined under a light microscope.

Drugs

[³ H]5-HT (specific activity=28 Ci/mmole) was obtained from New EnglandNuclear, Boston, Mass. All other chemicals were obtained from commercialsources and were of the highest grade purity available.

RESULTS

Isolation of a Genomic Clone Encoding a 5HT_(1E) Receptor

A human genomic placental library was screened with oligonucleotideprobes derived from transmembrane domains III, V and VI of the5-HT_(1D)β receptor gene. The hybridization of these probes with thelibrary was performed at low stringency and the result was theappearance of several hundred positive signals. Subsequently,approximately 350 of these clones were purified and categorized intovarious groups, based upon which of the three probes were responsiblefor the hybridization signal associated with a given clone. One group ofclones exhibited hybridization signals with both transmembrane domainprobes III and V. A number of these clones were subject to Southern blotanalysis and determined to be identical or overlapping clones. Arepresentative of this group, hp75d, was further characterized bynucleic acid sequence analysis and encoded what appeared to be a newserotonin receptor based upon its deduced amino acid sequence.

Nucleotide Sequence and Deduced Amino Acid Sequence of hp75d

DNA sequence information obtained from clone hp75d is shown in FIG. 1.An open reading frame extending from an ATG start codon at position 1 toa stop codon at position 1095 can encode a protein 365 amino acids inlength, having a relative molecular mass (M_(r)) of 41,633. A comparisonof this protein sequence with previously characterized neurotransmitterreceptors indicates that hp75d encodes a receptor which is a new memberof a family of molecules which span the lipid bilayer seven times andcouple to guanine nucleotide regulatory proteins (the G protein-coupledreceptor family). A variety of structural features which are invariantin this family were present including the aspartic acid residues oftransmembrane regions II and III, the DRY sequence at the end oftransmembrane region III, and the conserved proline residues oftransmembrane regions IV, V, VI and VII (Hartig et al. and referencestherein), were present in clone hp75d. A comparison of the transmembranehomology of hp75d to the other cloned serotonin receptors is shown inFIG. 2 and exhibits the following order of identity: 5-HT_(1D)β (65%),5-HT_(1D)β (64%), 5-HT_(1A) (52%), 5-HT_(1C) (40%) and 5-HT₂ (39%).

Receptor Expression in Transfected Mammalian Cells

Saturation analysis of membranes prepared from stably transfected Ltk⁻cells demonstrated that the receptor expressed was saturable and of highaffinity. Scatchard plot analysis by non-linear regression revealed a Kdof 10.3±1.2 nM (mean±S.E.M., n=7) and a Bmax consistent with a highlevel of expression, 10.9±2.6 picomoles/mg of protein (mean±S.E.M.,n=7). The percent specific binding determined at the measured Kd valuefor [³ H]5-HT was greater than 85% of total binding. Furthermore,evidence that the receptor is coupled to a G-protein was demonstrated bythe ability of Gpp(NH)p, a non-hydrolyzable analog of GTP, to inhibitthe specific binding of [³ H]5-HT (IC₅₀ =1885±556, n_(H) =0.87±0.04,I_(max) =26.4±5.6%).

Pharmacological analysis of the receptor was accomplished by testing theability of drugs from different chemical classes to displace [³ H]5-HTspecific binding (Table 1). Of the compounds investigated, 5-HT and5-hydroxylated tryptamine derivatives possessed the highest affinitywhich according to the classification system of Peroutka

    ______________________________________                                        COMPOUND         Ki (nm)                                                      ______________________________________                                        5-HT             10.9 ± 1.0                                                Lysergol         42.8 ± 5.3                                                Ergonovine       87.7 ± 7.6                                                Methylergonovine 89.4 ± 4.2                                                a-Methyl-5-HT    121 ± 13                                                  Methiothepin     194 ± 4                                                   1-Napthylpiperazine                                                                            207 ± 69                                                  Methysergide     228 ± 16                                                  Oxymetazoline    419 ± 49                                                  5-Methoxy-N,N-DMT                                                                              528 ± 32                                                  Ergotamine       599 ± 39                                                  2-Methyl-5-HT     817 ± 203                                                Yohimbine        1270 ± 233                                                Sumatriptan      2520 ± 135                                                Tryptamine       2559 ± 827                                                DOI              2970 ± 592                                                5-Methoxytryptamine                                                                             3151 ± 1041                                              8-OH-DPAT        3333 ± 310                                                Rauwolscine      3434 ± 102                                                Spiperone        5051 ± 689                                                TFMPP            6293 ± 259                                                5-CT             7875 ± 284                                                Ketanserin       >10,000                                                      Pindolol         >10,000                                                      Zacopride        >10,000                                                      LY-165163 (PAPP) >10,000                                                      DP-5-CT          >10,000                                                      ______________________________________                                    

and Snyder (1979) makes this site a member of the 5-HT₁ class.Interestingly, 5-CT possessed low affinity and, thus, discriminates thisreceptor from that of the 5-HT_(1D) receptor as well as other members ofthis class. Various ergoline compounds also bound with high affinityincluding agents which have potent hallucinogenic activity. Excludingmethiothepin and 1-napthylpiperazine (Ki values=194 and 207 nM,respectively), piperazine derivatives had low affinity and displayed Kivalues greater than 700 nM. Furthermore, rauwolfia alkaloids andserotonergic agents that possess high affinity for various subtypes ofreceptor within the serotonin family including ketanserin (5-HT₂),8-OH-DPAT (5-HT_(1A)), DOI (5- HT_(1C) /5-HT₂), spiperone (5-HT_(1A)/5-HT₂), pindolol (5-HT_(1A) /5-HT_(1B)) and zacopride (5-HT₃) had verypoor affinity. In all cases, the Hill Coefficients did not differsignificantly from unity. Taken together, the pharmacological profile ofthe 5-HT_(1E) receptor is unique and contrasts to that of other knownserotonin receptors. Accordingly, the probability of developingselective drugs for this receptor subtype is increased. Additionalsupporting evidence that the 5-HT_(1E) receptor is functionally coupledto a G-protein was obtained by testing the ability of 5-HT to inhibitforskolin-stimulated cAMP production in Y1 cells transfected with the5-HT_(1E) receptor. FIG. 4 demonstrates that the endogenous indoleamine,5-HT, produced a concentration-related decrease in forskolin-stimulatedcAMP production with an EC₅₀ of 23.1 nM. The maximum inhibition of cAMPproduction was 32%.

The tissue distribution of 5HT_(1E) receptor mRNA was detected using PCRtechnology on cDNA from tissue-derived total RNA. The mRNA localizationin human brain tissues demonstrates the presence of 5HT_(1E) in: frontalcortex, cerebellar cortex, temporal cortex, choroid plexus, hippocampus,brain stem and cortex. We have not identified an area of the brain whichdoes not contain 5HT_(1E). We conclude that the 5HT_(1E) mRNA isabundant in human brain. These findings suggest that the 5HT_(1E)receptor is not restricted to any one region of the brain and possiblycell type, but is expressed in numerous neuronal cell groups in manydistinct regions of the human brain, as has been described for 5HT_(1C).

The presence of 5HT_(1E) receptor mRNA in various regions of the humanbrain suggest that 5HT_(1E) may modulate a number of the central actionsattributed to serotonin. The abundance of 5HT_(1E) receptors (mRNA) inthe hippocampus may affect mood, behavior and hallucinogenesis. Agreater understanding of possible physiological roles of this receptorsubtype may be realized by the development of more specific 5HT_(1E)receptor drugs as well as physiological manipulations of 5HT_(1E)mRNAs/receptors.

The cellular localization of 5-HT_(1E) receptor mRNA was detected withdigoxigenin-11-dUTP labeled oligo probes employing in situhybridization(ISHH) technology. Digoxigenin-11-dUTP labeled oxytocin(OT)oligo probes were used as a positive control for the experiment sincethe distribution of OT neurons in the central nervous system is wellknown. OT cells were intensely stained in the Guinea Pig's hypothalamus.Tissue sections pre-treated with RNase A were used as a negativecontrol. Guinea pig brain sections were examined under a lightmicroscope. 5-HT_(1E) receptor mRNA was found in cells located in thefrontal cortex, piriform cortex, hippocampus (CA1, CA2and CA3), lateralseptal nucleus, triangular septal nucleus, septofimbrial nucleus and thebasal ganglia (caudate-putamen and globus pallidus). It was detected inthe amygdaloid complex, the bed nucleus stria terminalis and thehypothalamic area including anterior hypothalamus, periventricularnucleus, paraventricular nucleus (magnocellular and parvocellularpopulations), supraoptic nucleus which seemed to include bothvasopressin and oxytocin cell populations, and the lateral hypothalamus.The 1E receptor mRNA was also detected in the thalamic area includinganteroventral thalamic, anterodorsal thalamic, mediodorsal thalamic,ventrolateral thalamic, reticular thalamic paracentral thalamic,paratenial thalamic nuclei and the nucleus stria medullaris. Controlsections pre-treated with RNase A did not exhibit any staining pattern.

Experimental Discussion

The deduced amino acid sequence of hp75d was analyzed to uncoverrelationships between it and the other cloned serotonin receptorsequences. Although the homology within the membrane spanning domainswas greatest with the 5-HT_(1D)β receptor (FIG. 2), the nature of thisnewly cloned receptor could not be clearly predicted. The rational forthis ambiguity is the interpretation of the transmembrane domainhomology (approximately 65%) to the 5-HT_(1D)β and 5-HT_(1D)β receptorsubfamily. Closely related members of a "subfamily" of serotoninreceptors (i.e. "subtypes") generally share a common transmitter andalso have similar pharmacological profiles and physiological roles (forexample, 5-HT₂ and 5-HT_(1C) or 5-HT_(1D)α and 5-HT_(1D)β). Such"subtypes" display an amino acid identity of approximately 75-80% intheir transmembrane domains. Serotonin receptors which are not membersof the same "subfamily", but are members of the serotonin "family" (inwhich the receptors use the same neurotransmitter; i.e. 5-HT₂ and5-HT_(1D)α) generally show much lower transmembrane homology(approximately 45%). Such transmembrane amino acid homologies can,therefore, give insight into the relationship between receptors and beused as predictors of receptor pharmacology. According to this type ofanalysis, although the newly cloned receptor appears to be more relatedto the 5-HT_(1D) subfamily, it is likely to be in a subfamily distinctfrom all the other serotonin receptors.

The present pharmacological evidence substantiates the existence of anovel serotonin receptor in the human brain as first suggested byLeonhardt et al. (1989). Comparison of the pharmacological profileobserved in native cortical membranes to that revealed for the cloned5-HT_(1E) receptor yielded a correlation coefficient of 0.987 (FIG. 3).Indeed, there were some differences in measured affinity constants butthe relative values of selected drugs performed in both studies wassimilar. In this regard, a close examination of the Scatchard plotanalysis performed in the study by Leonhardt et al. (1989) reveals thatthe reported Kd value may have been underestimated since the radioligandconcentrations did not exceed 10 nM. In order to have accuratelydetermined the dissociation constant, the radioligand concentrationshould have been extended to at least 50 nM or 10 times the estimated Kd(Yamamura et al., 1985). The initial study, however, was limited intissue supply and lacked a comprehensive pharmacologicalcharacterization. The cloning of the 5-HT_(1E) site will now allow moreextensive investigations into the nature of this unique receptor.

The structure-activity relationships observed in the present studysuggest that there are important requirements for high affinity bindingto the 5-HT_(1E) receptor. Substitution or removal of the 5-hydroxygroup on serotonin decreases the affinity 300 fold for the receptor(egs., tryptamine, 5-methoxytryptamine and 5-carboxyamidotryptamine).Additionally, 2-methylation and α-methylation of 5-HT essentiallyabolishes its affinity for the 5-HT_(1E) site. In contrast to thesesubstitutions, N,N -dimethylation of the aliphatic side chain of theindole ring increases the affinity approximately 20 fold (unpublishedobservations). Basic structural requirements of the ergoline derivativesdemonstrate that N-methylation of the indole ring decreases affinity asdoes bulky substitutions. Furthermore, piperazine derivatives are notbound at high affinity.

Notably, the application of the human 5-HT_(1E) receptor clone topharmaceutical research can lead to new drug design and development. Thelocalization of this receptor in the cerebral cortex (Leonhardt et al.,1989) and parts of the basal ganglia such as the putamen and globuspallidus (Lowther et al., 1991) suggests a putative link to limbic,cognitive and/or motor function (Nieuwenhuys et al., 1988; Kandel andSchwartz, 1985) and, thus, may be involved in such abnormal conditionsas dementia, Parkinson's disease, feeding disorders, anxiety andschizophrenia. Notably, the ergot compounds that possess affinity forthis site have been demonstrated to affect these type of behaviors inhumans as well as animals (Wilkinson and Dourish, 1991). In relation tothis, it appears that the 5-HT_(1E) binding site is also present in ratand bovine brain (Leonhardt et al., 1989) as well as guinea-pig, rabbitand dog where the data was initially interpreted as evidence forsubtypes of the 5-HT_(1D) receptor (Middlemiss, 1990). Nonetheless, itmust be taken into consideration that this novel site can possibly leadto selective drug therapy devoid of side effects. In regard to this,serotonin uptake blockers are effective in treating neuropsychiatricdisorders such as depression and obsessive-compulsive illness (Blier etal., 1987; Asberg et al., 1986; Insel et al., 1985). However, theseagents have side effects and, in fact, the mechanism of action for thesecompounds are not linked to any particular serotonergic receptor. Thepossibility that agents selective for the 5-HT_(1E) receptor may haveclinical utility as antidepressants, for example, without the sideeffects attributed to current treatment modalities can have significantimplications for drug therapy. Furthermore, it should be noted thatergoline derivatives have had clinical usefulness as drugs capable ofrelieving migraines and, thus, the involvement of the 5-HT_(1E) receptorin this disorder deserves future attention. Ultimately, in depthinvestigations into the localization of the 5-HT_(1E) receptor in brainand peripheral tissue will target new sites that can lead to specificfunctional roles for this serotonergic receptor.

Another consideration for therapeutic application of this site may berelated to the treatment of feeding disorders such as obesity, bulimianervosa and/or anorexia nervosa. The involvement of serotonin andfeeding behavior has received much attention during the last decade. Itis now known that many of the identified and well-characterizedserotonergic receptors are capable of modulating feeding (Blundell andLawton, 1990). Notably, serotonin uptake blockers which have been usedto treat feeding disorders act nonselectively and as such haveside-effect potential (Jimerson et al., 1990). Although many differentserotonergic receptors are involved in feeding, the search for the onesite that can be exploited for selective drug development has yet to befound. There is no doubt that interest exists in finding drugs thatinteract with the serotonin system for the treatment of feedingdisorders (Cooper, 1989).

Thus, the pharmacological profile of the cloned human 5-HT_(1E) receptoris unique and contrasts to other known serotonergic receptors. Theutility of this site expressed in a cellular system and, thus, isolatedfor study will create excellent opportunities in drug developmentdirected towards a novel serotonergic receptor that may have wide-rangeimplications for drug therapy. Indeed, the potential therapeuticapplications may extend to neuropsychiatric disorders includingdepression, anxiety, schizophrenia, dementia and obsessive-compulsiveillness as well as obesity and migraine. The localization of 5-HT_(1E)receptor mRNA by in situ hybridization makes it possible to predict itsphysiological and pathological functions. 5-HT_(1E) receptor mRNA isdetected in the limbic structures, such as the hippocampus, septalnuclei, piriform cortex (olfactory system), amygdaloid complex and thebed nucleus stria terminalis. The olfactory system sends afferent fibersto the hippocampus through the subiculum, and to the amygdaloid complex(Kupfermann,1985). In turn the outputs of the hippocampus project to theseptal area and hypothalamus while the amygdaloid complex projects tothe hypothalamus via the stria terminalis (Kupfermann, 1985). Theinvolvement of the limbic system in emotional behavior (e.g., fear,pleasure,sexual activities) and memory is well known (Kupfermann,1985).Therefore, the finding of 5-HT_(1E) receptor mRNA in these structuresindicates a potential role in neuropsychiatric disorders such asdepression, obsessive-compulsive illness, anxiety, schizophrenia anddementia.

The hypothalamus regulates the body adjustments to the external andinternal environments. It can control hunger (the ventromedial nucleusand the lateral hypothalamus), endocrine functions (e.g. the supraopticnucleus, the paraventricular nucleus and the periventricular nucleus),affective (emotional) behavior (the ventromedial and dorsomedial nuclei)and the activity of the visceral nervous system (the anteriorhypothalamus) (Diamond et al., 1985). The discovery of 5-HT_(1E)receptor mRNA in these nuclei indicates physiological and pathologicalroles of this receptor subtype in cardiovascular, gastrointestinal,endocrine, neurological and psychiatric systems.

The thalamus is a relay station where the sensory and motor-relatedpathways passing up the brain stem synapse before proceeding on to thecerebral cortex for more elaborate integration and analysis. The5-HT_(1E) receptor mRNA was found in the anterior thalamic nucleus whichreceives the input from the mammillothalamic tract and sends fibers tothe cingulate gyrus. Thus, this nucleus is an important part of thecircuit connecting the hypothalamus, the thalamus, and the limbic lobe(Diamond et al., 1985). The 5-HT_(1E) receptor mRNA was also found inthe mediodorsal thalamic nucleus which is involved in emotional behaviorand in the ventral lateral thalamic nucleus which connects the basalganglia and premotor area. Furthermore, 5-HT_(1E) receptor mRNA wasfound in the nucleus reticularis thalami through which pass most of thethalamocortical and corticothalamic fibers. The axons of corticothalamicand thalamocortical neurons provide collaterals for synapse withreticular cells, exerting a facilitatory effect, while the axons of theGABA-rich reticularis cells project back on the specific thalamicneurons, continuously modulating (by inhibition) the ascending flow ofthalamocortical impulses (Diamond et al., 1985). The reticularis neuronsplay a significant role in the interactions between the thalamus and thefrontal cortex. In addition, the 5-HT_(1E) receptor mRNA was alsovisualized in the basal ganglia including the caudate-putamen and globuspallidus both of which control motor activity (Alheid et al., 1990).These findings indicate important roles of the 5-HT_(1E) receptors inemotional behavior, sensation (e.g., pain) and motor activity.

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Glennon, R. A.: Serotonin receptors: Clinical implications. Neurosci.Biobehav. Rev. 14:35-47, 1990.

Green, A. R.: Neuropharmacology of serotonin. Oxford: Oxford UniversityPress, 1985.

Hamon, M., Lanfumey, L., El Mestikawy, S., Boni, C., Miquel, M.-C.,Bolanos, F., Schechter, L. and Gozlan, H.: The main features of central5-HT1 receptors. Neuropsychopharmacol. 3(5/6):349-360, 1990.

Hartig, P. R., Kao, H.-T., Macchi, M., Adham, N., Zgombick, J.,Weinshank, R. and Branchek, T.: The molecular biology of serotoninreceptors: An overview. Neuropsychopharmacol. 3(5/6):335-347, 1990.

Insel, T. R., Mueller, E. A., Alterman, I., Linnoila, M. and Murphy, D.L.: Obsessive-compulsive disorder and serotonin: Is there a connection?Biol. Psychiat. 20:1174-1188, 1985.

Jimerson, D. C., Lesem, M. D., Hegg, A. P. and Brewerton, T. D.:Serotonin in human eating disorders. Ann. N.Y. Acad. Sci. 600:532-544,1990.

Julius, D., MacDermott, A. B., Axel, R. and Jessell, T. M.: Molecularcharacterization of a functional cDNA encoding the serotonin 1Creceptor. Science 241:558-564, 1988.

Kandel, E. R. and Schwartz, J. H.: Principles of neuroscience. New York:Elsevier Publishing Co., 1985.

Kupfermann, I.: Hypothalamus and limbic system. In: Principles ofneuroscience. Eds: Kandel, E. R. and Schwartz, J. H., New York: ElsevierPublishing Co.,1985

Leonhardt, S., Herrick-Davis, K. and Titeler, M.: Detection of a novelserotonin receptor subtype (5-HT1E) in human brain: Interaction with aGTP-binding protein. J. Neurochem. 53(2):465-471, 1989.

Lowther, S., De Paermentier, F., Crompton, M. R., Katona, C. L. E. andHorton, R. W.: 5HT_(1D) and 5HT_(1E) binding sites in depression: Apost-mortem study in suicide victims. Serotonin 1991-5-Hydroxytryptamine--CNS Receptors and Brain Function, Birmingham, UK,Jul. 14-17, 1991, p. 175, Abstract #P. 145.

Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982.

Middlemiss, D. N., Suman-Chauhan, N., Smith, S. M., Picton, C., Shaw, D.and Bevan, Y.: Subpopulations of 5-HT1D recognition sites in guinea-pig,rabbit, dog and human cortex. The Second IUPHAR Satellite Meeting onSerotonin, Basel, Switzerland, Jul. 11-13, 1990, Abstract #P-30.

Miller, J., and R. N. Germain. Efficient cell surface expression ofclass II MHC molecules in the absence of associated invariant chain.J.Exp.Med. 164:1478-1489, 1986.

Nieuwenhuys, R., Voogd, J. and van Huijzen, C.: The human nervoussystem: A synopsis and atlas. New York: Springer Verlag, 1988.

Osborne, N. N. and Hamon, M.: Neuronal serotonin. Chichester: John Wileyand Sons, Inc., 1988.

Peroutka, S. J.: Serotonin receptor subtypes: Basic and clinicalaspects. New York: Wiley-Liss, Inc., 1991.

Peroutka, S. J. and Snyder, S. H.: Multiple serotonin receptors,differential binding of [³ H]5-hydroxytryptamine, [³ H]lysergic aciddiethylamide and [³ H]spiroperidol. Mol. Pharmacol. 16:687-699, 1979.

Pritchett, D. B., Bach, A. W. J., Wozny, M., Taleb, O., Dal Toso, R.,Shih, J. and Seeburg, P. H.: Structure and functional expression ofcloned rat serotonin 5-HT2 receptor. EMBO J. 7:4135-4140, 1988.

Rapport, M. M., Green, A. A. and Page, I. H.: Purification of thesubstance which is responsible for vasoconstrictor activity of serum.Fed. Proc. 6:184, 1947.

Rapport, M. M.: Serum vasoconstrictor (serotonin) V. Presence ofcreatinine in the complex. A proposed structure of the vasoconstrictorprinciple. J. Biol. Chem. 180:961-969, 1949.

Sanger, S. DNA sequencing with chain-terminating inhibitors. Proc.Natl.Acad. Sci.USA 74:5463-5467, 1977.

Sanders-Bush, E.: The Serotonin Receptors. Clifton, N.J.: Humana Press,1988.

Southern, E. M. Detection of specific sequences among DNA fragmentsseparated by gel electrophoresis. J.Mol.Biol. 98:503-505, 1975.

Wilkinson, L. O. and Dourish, C. T.: Serotonin and animal behavior. In:Serotonin receptor subtypes: Basic and clinical aspects. (ed. Peroutka,S. J.) New York:Wiley-Liss, Inc., pgs. 147-210, 1991.

Yamamura, H. I., Enna, S. J. and Kuhar, M. J.: Neurotransmitter receptorbinding. New York: Raven Press, 1985.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2463 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                          (v) FRAGMENT TYPE: N-terminal                                                 (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: human placental genomic                                          (B) CLONE: hp75d                                                              (viii) POSITION IN GENOME:                                                    (C) UNITS: bp                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 736..1830                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 736..1830                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATATACATCATGGAATACTATGCAGCCCCCCCCAAGGATGCGTTCCATGTCCTTTGCAGG60                GACATGGATGAGTTGCAAACCATATTCTCAACAAACTAACACAGCCACAGAAAACCAAAC120               ACCACATGCTCTCAC TCACGAGTGGAGTTGAACAATGAGAACACATGGCACAGGGCCGGG180              AACATGACACACCAGGGCCTGTTGGGGGGTGGAGGGCTAGGGGAGGGATGGCATTAGGAG240               AAGTACCTAATGTAGATGATTGGTTGTTGGGTGCAGCAAACCACCATGGCACATGTATA C300              CTATGTAGCAAACCTGCAAGTTCTGCACATGTATCCCAGGACTTAAAGTATAATTTAAAA360               AAAAACAGTTTGAAAACTTCCCTGAAGTAAAAAAAGTATCCTTTGAGGAACAATGTAACG420               ATGAGCTCAAGTTCCACAGGAAAGAGAAAATTA AAATTTATAAAGAATTTATAAATATCA480              AACTATTTTCATGTTTTCCAGGAAAAGTGTGGCTTTCTCATTCATTAACCAATAGCATAA540               TATTTTCCAGGAACCTTCACTCAGAAGAAATGCTGTGGCCCTTCCCTTTACCAACAGAAA600               ATGGAACA CAAGAGACCACATAGCTGAACAAATTATAGCCTCCTTACAAGTGAGAAACCT660              TCGAGGCTACATAGTTTTCAGCCAAAGGAAAATAACCAACAGCTTCTCCACAGTGTAGAC720               TGAAACAAGGGAAACATGAACATCACAAACTGTACCACAGAGGCC AGCATG771                       MetAsnIleThrAsnCysThrThrGluAlaSerMet                                          1510                                                                          GCTATAAGACCCAAGACCATCACTGAGAAGATGCTCATTTG CATGACT819                          AlaIleArgProLysThrIleThrGluLysMetLeuIleCysMetThr                              152025                                                                        CTGGTGGTCATCACCACCCTCACCACGTTGCTGAACTTGGCTGT GATC867                          LeuValValIleThrThrLeuThrThrLeuLeuAsnLeuAlaValIle                              303540                                                                        ATGGCTATTGGCACCACCAAGAAGCTCCACCAGCCTGCCAACTACCTA 915                          MetAlaIleGlyThrThrLysLysLeuHisGlnProAlaAsnTyrLeu                              45505560                                                                      ATCTGTTCTCTGGCCGTGACGGACCTCCTGGTGGCAGTGCTCGT CATG963                          IleCysSerLeuAlaValThrAspLeuLeuValAlaValLeuValMet                              657075                                                                        CCCCTGAGCATCATCTACATTGTCATGGATCGCTGGAAGCT TGGGTAC1011                         ProLeuSerIleIleTyrIleValMetAspArgTrpLysLeuGlyTyr                              808590                                                                        TTCCTCTGTGAGGTGTGGCTGAGTGTGGACATGACCTGCTG CACCTGC1059                         PheLeuCysGluValTrpLeuSerValAspMetThrCysCysThrCys                              95100105                                                                      TCCATCCTCCACCTCTGTGTCATTGCCCTGGACAGGTACTGGGC CATC1107                         SerIleLeuHisLeuCysValIleAlaLeuAspArgTyrTrpAlaIle                              110115120                                                                     ACCAATGCTATTGAATACGCCAGGAAGAGGACGGCCAAGAGGGCCGCG 1155                         ThrAsnAlaIleGluTyrAlaArgLysArgThrAlaLysArgAlaAla                              125130135140                                                                  CTGATGATCCTTACCGTCTGGACCATCTCCATTTTCATCTCCAT GCCC1203                         LeuMetIleLeuThrValTrpThrIleSerIlePheIleSerMetPro                              145150155                                                                     CCTCTGTTCTGGAGAAGCCACCGCCGCCTAAGCCCTCCCCC TAGTCAG1251                         ProLeuPheTrpArgSerHisArgArgLeuSerProProProSerGln                              160165170                                                                     TGCACCATCCAGCACGACCATGTTATCTACACCATTTACTC CACGCTG1299                         CysThrIleGlnHisAspHisValIleTyrThrIleTyrSerThrLeu                              175180185                                                                     GGTGCGTTTTATATCCCCTTGACTTTGATACTGATTCTCTATTA CCGG1347                         GlyAlaPheTyrIleProLeuThrLeuIleLeuIleLeuTyrTyrArg                              190195200                                                                     ATTTACCACGCGGCCAAGAGCCTTTACCAGAAAAGGGGATCAAGTCGG 1395                         IleTyrHisAlaAlaLysSerLeuTyrGlnLysArgGlySerSerArg                              205210215220                                                                  CACTTAAGCAACAGAAGCACAGATAGCCAGAATTCTTTTGCAAG TTGT1443                         HisLeuSerAsnArgSerThrAspSerGlnAsnSerPheAlaSerCys                              225230235                                                                     AAACTTACACAGACTTTCTGTGTGTCTGACTTCTCCACCTC AGACCCT1491                         LysLeuThrGlnThrPheCysValSerAspPheSerThrSerAspPro                              240245250                                                                     ACCACAGAGTTTGAAAAGTTCCATGCCTCCATCAGGATCCC CCCCTTC1539                         ThrThrGluPheGluLysPheHisAlaSerIleArgIleProProPhe                              255260265                                                                     GACAATGATCTAGATCACCCAGGAGAACGTCAGCAGATCTCTAG CACC1587                         AspAsnAspLeuAspHisProGlyGluArgGlnGlnIleSerSerThr                              270275280                                                                     AGGGAACGGAAGGCAGCACGCATCCTGGGGCTGATTCTGGGTGCATTC 1635                         ArgGluArgLysAlaAlaArgIleLeuGlyLeuIleLeuGlyAlaPhe                              285290295300                                                                  ATTTTATCCTGGCTGCCATTTTTCATCAAAGAGTTGATTGTGGG TCTG1683                         IleLeuSerTrpLeuProPhePheIleLysGluLeuIleValGlyLeu                              305310315                                                                     AGCATCTACACCGTGTCCTCGGAAGTGGCCGACTTTCTGAC GTGGCTC1731                         SerIleTyrThrValSerSerGluValAlaAspPheLeuThrTrpLeu                              320325330                                                                     GGTTATGTGAATTCTCTGATCAACCCTCTGCTCTATACGAG TTTTAAT1779                         GlyTyrValAsnSerLeuIleAsnProLeuLeuTyrThrSerPheAsn                              335340345                                                                     GAAGACTTTAAGCTGGCTTTTAAAAAGCTCATTAGATGCCGAGA GCAT1827                         GluAspPheLysLeuAlaPheLysLysLeuIleArgCysArgGluHis                              350355360                                                                     ACTTAGACTGTAAAAAGCTAAAAGGCACGACTTTTTCCAGAGCCTCATGAGTG 1880                    Thr                                                                           365                                                                           GATGGGGGTAAGGGGTGCAACTTATTAATTCTTGAACATACTTGGTTCAGGAGAGTTTGT1940              AAGTATGTGTGGTCTTGTTTCCTTGTTTGTTTGTTTGTTTTGTTCTGTTTTGTTTGAGGA2000              TTGTTATTTGGCCTCCTGTTTTCT ACCTCTGGTCTTATCTGTGATACATAATTTCAAATA2060             AACATTATCATACAAAAACAGAAATTTTGCCAGAAGTAATAATAAGATGAAATACTAAAT2120              ACCTTTTATGGGTTTTTTTTTTTTAGCCATTTCAGTTACCCTGCAATTAAAGAATGCCAA2180              AAATATCTTTATTTGCAGAATTTCTTATTACTTATAAATTAAATACCTGATAATGCCCTC2240              CATGGCATTAAATCTGAGATTATGGCTCTATCTGCGTACATATTCCAGTGGGAATTGCAT2300              GACTACATAAAGAATTAAAAGAAAGTGATGTGCTGTCATCTA CGGCTTGCGACCTGAGCT2360             AAAGTCGGGGGCTGTAGCACTGTGACTACGTAGCCTATCATTTCAGGTAAAAATAGTACA2420              GCTGGCTTGTCTTGTTAGTTCATGATTAAATAAACTTCTCTTT2463                               (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 365 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAsnIleThrAsnCysThrThrGluAlaSerMetAlaIleArgPro                              15 1015                                                                       LysThrIleThrGluLysMetLeuIleCysMetThrLeuValValIle                              202530                                                                        ThrThrLeuThrThrLeuLeuAsnLeuAlaValIle MetAlaIleGly                             354045                                                                        ThrThrLysLysLeuHisGlnProAlaAsnTyrLeuIleCysSerLeu                              505560                                                                        AlaValTh rAspLeuLeuValAlaValLeuValMetProLeuSerIle                             65707580                                                                      IleTyrIleValMetAspArgTrpLysLeuGlyTyrPheLeuCysGlu                               859095                                                                       ValTrpLeuSerValAspMetThrCysCysThrCysSerIleLeuHis                              100105110                                                                     LeuCysValIleAlaLeu AspArgTyrTrpAlaIleThrAsnAlaIle                             115120125                                                                     GluTyrAlaArgLysArgThrAlaLysArgAlaAlaLeuMetIleLeu                              130135 140                                                                    ThrValTrpThrIleSerIlePheIleSerMetProProLeuPheTrp                              145150155160                                                                  ArgSerHisArgArgLeuSerProProProSerGlnCys ThrIleGln                             165170175                                                                     HisAspHisValIleTyrThrIleTyrSerThrLeuGlyAlaPheTyr                              180185190                                                                     IleProLeuThrLeuIleLeuIleLeuTyrTyrArgIleTyrHisAla                              195200205                                                                     AlaLysSerLeuTyrGlnLysArgGlySerSerArgHisLeuSerAsn                              210 215220                                                                    ArgSerThrAspSerGlnAsnSerPheAlaSerCysLysLeuThrGln                              225230235240                                                                  ThrPheCysValSerAspPhe SerThrSerAspProThrThrGluPhe                             245250255                                                                     GluLysPheHisAlaSerIleArgIleProProPheAspAsnAspLeu                              260 265270                                                                    AspHisProGlyGluArgGlnGlnIleSerSerThrArgGluArgLys                              275280285                                                                     AlaAlaArgIleLeuGlyLeuIleLeuGlyAlaPheIle LeuSerTrp                             290295300                                                                     LeuProPhePheIleLysGluLeuIleValGlyLeuSerIleTyrThr                              305310315320                                                                  Va lSerSerGluValAlaAspPheLeuThrTrpLeuGlyTyrValAsn                             325330335                                                                     SerLeuIleAsnProLeuLeuTyrThrSerPheAsnGluAspPheLys                               340345350                                                                    LeuAlaPheLysLysLeuIleArgCysArgGluHisThr                                       355360365                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetAspValLeuSerProGlyGlnGlyAsnAsnThrThrSerP roPro                             151015                                                                        AlaProPheGluThrGlyGlyAsnThrThrGlyIleSerAspValThr                              2025 30                                                                       ValSerTyrGlnValIleThrSerLeuLeuLeuGlyThrLeuIlePhe                              354045                                                                        CysAlaValLeuGlyAsnAlaCysValValAlaAlaIleAla LeuGlu                             505560                                                                        ArgSerLeuGlnAsnValAlaAsnTyrLeuIleGlySerLeuAlaVal                              657075 80                                                                     ThrAspLeuMetValSerValLeuValLeuProMetAlaAlaLeuTyr                              859095                                                                        GlnValLeuAsnLysTrpThrLeuGlyGlnValThrC ysAspLeuPhe                             100105110                                                                     IleAlaLeuAspValLeuCysCysThrSerSerIleLeuHisLeuCys                              115120 125                                                                    AlaIleAlaLeuAspArgTyrTrpAlaIleThrAspProIleAspTyr                              130135140                                                                     ValAsnLysArgThrProArgArgAlaAlaAlaLeuIleSer LeuThr                             145150155160                                                                  TrpLeuIleGlyPheLeuIleSerIleProProMetLeuGlyTrpArg                              165170 175                                                                    ThrProGluAspArgSerAspProAspAlaCysThrIleSerLysAsp                              180185190                                                                     HisGlyTyrThrIleTyrSerThrPheGly AlaPheTyrIleProLeu                             195200205                                                                     LeuLeuMetLeuValLeuTyrGlyArgIlePheArgAlaAlaArgPhe                              210215 220                                                                    ArgIleArgLysThrValLysLysValGluLysThrGlyAlaAspThr                              225230235240                                                                  ArgHisGlyAlaSerProAlaProGlnPr oLysLysSerValAsnGly                             245250255                                                                     GluSerGlySerArgAsnTrpArgLeuGlyValGluSerLysAlaGly                              260 265270                                                                    GlyAlaLeuCysAlaAsnGlyAlaValArgGlnGlyAspAspGlyAla                              275280285                                                                     AlaLeuGluValIleGluValHisA rgValGlyAsnSerLysGluHis                             290295300                                                                     LeuProLeuProSerGluAlaGlyProThrProCysAlaProAlaSer                              305310 315320                                                                 PheGluArgLysAsnGluArgAsnAlaGluAlaLysArgLysMetAla                              325330335                                                                     LeuAlaArgGluArgLys ThrValLysThrLeuGlyIleIleMetGly                             340345350                                                                     ThrPheIleLeuCysTrpLeuProPhePheIleValAlaLeuValLeu                              355 360365                                                                    ProPheCysGluSerSerCysHisMetProThrLeuLeuGlyAlaIle                              370375380                                                                     IleAsnTrpLeuGlyTyrSerAsn SerLeuLeuAsnProValIleTyr                             385390395400                                                                  AlaTyrPheAsnLysAspPheGlnAsnAlaPheLysLysIleIleLys                              4 05410415                                                                    CysLeuPheCysArgGln                                                            420                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 460 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetValAsnLeuGlyAsnAlaValArgSerLeuLeuMetHisLeuIle                              1510 15                                                                       GlyLeuLeuValTrpGlnPheAspIleSerIleSerProValAlaAla                              202530                                                                        IleValThrAspThrPheAsnSerSerAspGlyG lyArgLeuPheGln                             354045                                                                        PheProAspGlyValGlnAsnTrpProAlaLeuSerIleValValIle                              5055 60                                                                       IleIleMetThrIleGlyGlyAsnIleLeuValIleMetAlaValSer                              65707580                                                                      MetGluLysLysLeuHisAsnAlaThrAsnTyrPhe LeuMetSerLeu                             859095                                                                        AlaIleAlaAspMetLeuValGlyLeuLeuValMetProLeuSerLeu                              100105 110                                                                    LeuAlaIleLeuTyrAspTyrValTrpProLeuProArgTyrLeuCys                              115120125                                                                     ProValTrpIleSerLeuAspValLeuPheSer ThrAlaSerIleMet                             130135140                                                                     HisLeuCysAlaIleSerLeuAspArgTyrValAlaIleArgAsnPro                              145150155 160                                                                 IleGluHisSerArgPheAsnSerArgThrLysAlaIleMetLysIle                              165170175                                                                     AlaIleValTrpAlaIleSerIleGl yValSerValProIleProVal                             180185190                                                                     IleGlyLeuArgAspGluSerLysValPheValAsnAsnThrThrCys                              195 200205                                                                    ValLeuAsnAspProAsnPheValLeuIleGlySerPheValAlaPhe                              210215220                                                                     PheIleProLeuThrIleMetValIleThrT yrPheLeuThrIleTyr                             225230235240                                                                  ValLeuArgArgGlnThrLeuMetLeuLeuArgGlyHisThrGluGlu                              245 250255                                                                    GluLeuAlaAsnMetSerLeuAsnPheLeuAsnCysCysCysLysLys                              260265270                                                                     AsnGlyGlyGluGluGlu AsnAlaProAsnProAsnProAspGlnLys                             275280285                                                                     ProArgArgLysLysLysGluLysArgProArgGlyThrMetGlnAla                              290 295300                                                                    IleAsnAsnGluLysLysAlaSerLysValLeuGlyIleValPhePhe                              305310315320                                                                  ValPheLeuIleMetTrp CysProPhePheIleThrAsnIleLeuSer                             325330335                                                                     ValLeuCysGlyLysAlaCysAsnGlnLysLeuMetGluLysLeuLeu                              3 40345350                                                                    AsnValPheValTrpIleGlyTyrValCysSerGlyIleAsnProLeu                              355360365                                                                     ValTyrThrLeuPh eAsnLysIleTyrArgArgAlaPheSerLysTyr                             370375380                                                                     LeuArgCysAspTyrLysProAspLysLysProProValArgGlnIle                              385 390395400                                                                 ProArgValAlaAlaThrAlaLeuSerGlyArgGluLeuAsnValAsn                              405410415                                                                     IleTyrA rgHisThrAsnGluArgValAlaArgLysAlaAsnAspPro                             420425430                                                                     GluProGlyIleGluMetGlnValGluAsnLeuGluLeuProValAsn                               435440445                                                                    ProSerAsnValValSerGluArgIleSerSerVal                                          450455460                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 375 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetSerProLeuAsnGlnSerAlaGluGlyLeuProGlnGlu AlaSer                             151015                                                                        AsnArgSerLeuAsnAlaThrGluThrSerGluAlaTrpAspProArg                              2025 30                                                                       ThrLeuGlnAlaLeuLysIleSerLeuProValLeuLeuSerValIle                              354045                                                                        ThrLeuAlaThrValLeuSerAsnAlaPheValLeuThrT hrIleLeu                             505560                                                                        LeuThrArgLysLeuHisThrProAlaAsnTyrLeuIleGlySerLeu                              657075 80                                                                     AlaThrThrAspLeuLeuValSerIleLeuValMetProIleSerMet                              859095                                                                        AlaTyrThrIleThrHisThrTrpAsnPheGlyGln IleLeuCysAsp                             100105110                                                                     IleTrpLeuSerSerAspIleThrCysCysThrAlaSerIleLeuHis                              115120 125                                                                    LeuCysValIleAlaLeuAspArgTyrTrpAlaIleThrAspAlaLeu                              130135140                                                                     GluTyrSerLysArgArgThrAlaGlyHisAlaAlaThrMe tIleAla                             145150155160                                                                  IleValTrpAlaIleSerIleCysIleSerIleProProLeuPheTrp                              165170 175                                                                    ArgGlnGluLysAlaGlnGluGluMetSerAspCysLeuValAsnThr                              180185190                                                                     SerGlnIleSerTyrThrIleTyrSerT hrCysGlyAlaPheTyrIle                             195200205                                                                     ProSerValLeuLeuIleIleLeuTyrGlyArgIleTyrArgAlaAla                              210215 220                                                                    ArgAsnArgIleLeuAsnProProSerLeuSerGlyLysArgPheThr                              225230235240                                                                  ThrAlaHisLeuIleThrGlySerAla GlySerValCysSerLeuAsn                             245250255                                                                     SerSerLeuHisGluGlyHisSerHisSerAlaGlySerProLeuPhe                              260 265270                                                                    PheAsnHisValLysIleLysLeuAlaAspSerAlaLeuGluArgLys                              275280285                                                                     ArgIleSerAlaAlaArgGluArg LysAlaThrLysIleLeuGlyIle                             290295300                                                                     IleLeuGlyAlaPheIleIleCysTrpLeuProPhePheValValSer                              305310 315320                                                                 LeuValLeuProIleCysArgAspSerCysTrpIleHisProGlyLeu                              325330335                                                                     PheAspPhePheThrTr pLeuGlyTyrLeuAsnSerLeuIleAsnPro                             340345350                                                                     IleIleTyrThrValPheAsnGluGluPheArgGlnAlaPheGlnLys                              355 360365                                                                    IleValProPheArgLysAla                                                         370375                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 398 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                      (D) TOPOLOGY: unknown                                                        (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluGluProGlyAlaGlnCysAlaProProAlaProAlaGlySer                              151 015                                                                       GluThrTrpValProGlnAlaAsnLeuSerSerAlaProSerGlnAsn                              202530                                                                        CysSerAlaLysAspTyrIleTyrGln AspSerIleSerLeuProTrp                             354045                                                                        LysValLeuLeuValMetLeuLeuAlaLeuIleThrLeuAlaThrThr                              5055 60                                                                       LeuSerAsnAlaPheValIleAlaThrValTyrArgThrArgLysLeu                              65707580                                                                      HisThrProAlaAsnTyrLeuIleAlaSe rLeuAspValThrAspLeu                             859095                                                                        LeuValSerIleLeuValIleProIleSerThrMetTyrThrValThr                              100 105110                                                                    AspArgTrpThrLeuSerGlnValValCysAspPheTrpLeuSerSer                              115120125                                                                     AspIleThrCysCysThrAlaSerIl eLeuHisLeuCysValIleAla                             130135140                                                                     LeuAspArgTyrTrpAlaIleThrAspAlaValGluTyrSerAlaLys                              145150 155160                                                                 ArgThrProLysArgAlaAlaValMetIleAlaLeuValTrpValPhe                              165170175                                                                     SerIleSerIleSerLeuP roProPhePheTrpArgGlnAlaLysAla                             180185190                                                                     GluGluGluValSerGluCysValValAsnThrAspHisIleLeuTyr                              195 200205                                                                    ThrValTyrSerThrValGlyAlaPheTyrPheProThrLeuLeuLeu                              210215220                                                                     IleAlaLeuTyrGlyArgIleTyr ValGluAlaArgSerArgIleLeu                             225230235240                                                                  LysGlnThrProAsnArgThrGlyLysArgLeuThrArgAlaGlnLeu                              24 5250255                                                                    IleThrAspSerProGlySerThrSerSerValThrSerIleAsnSer                              260265270                                                                     ArgValProAsp ValProSerGluSerGlySerProValTyrValAsn                             275280285                                                                     GlnValLysValArgValSerAspAlaLeuLeuGluLysLysLysLeu                              290 295300                                                                    MetAlaAlaArgGluArgLysAlaThrLysThrLeuGlyIleIleLeu                              305310315320                                                                  GlyAlaPheIl eValCysTrpLeuProPhePheIleIleSerLeuVal                             325330335                                                                     MetProIleCysLysAspAlaCysTrpPheHisLeuAlaIlePheAsp                               340345350                                                                    PhePheThrTrpLeuGlyTyrLeuAsnSerLeuIleAsnProIleIle                              355360365                                                                     TyrThrM etSerAsnGluAspPheLysGlnAlaPheHisLysLeuIle                             370375380                                                                     ArgLeuSerAlaGlnValAspLeuProPheAlaValGlyPro                                    385 390395                                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 471 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetAspIleLeuCysGluGluAsnThrSerLeuSerSerThrThrAsn                              151015                                                                        SerLeuMetGlnLeuAsnAspAspThrArgLeuTyrSer AsnAspPhe                             202530                                                                        AsnSerGlyGluAlaAsnThrSerAspAlaPheAsnTrpThrValAsp                              3540 45                                                                       SerGluAsnArgThrAsnLeuSerCysGluGlyCysLeuSerProSer                              505560                                                                        CysLeuSerLeuLeuHisLeuGlnGluLysAsnTrpSerAlaLeuLe u                             65707580                                                                      ThrAlaValValIleIleLeuThrIleAlaGlyAsnIleLeuValIle                              8590 95                                                                       MetAlaValSerLeuGluLysLysLeuGlnAsnAlaThrAsnTyrPhe                              100105110                                                                     LeuMetSerLeuAlaIleAlaAspMetLeuLeuGly PheLeuValMet                             115120125                                                                     ProValSerMetLeuThrIleLeuTyrGlyTyrArgTrpProLeuPro                              1301351 40                                                                    SerLysLeuCysAlaValTrpIleTyrLeuAspValLeuPheSerThr                              145150155160                                                                  AlaSerIleMetHisLeuCysAlaIleSerLeuAs pArgTyrValAla                             165170175                                                                     IleGlnAsnProIleHisHisSerArgPheAsnSerArgThrLysAla                              180185 190                                                                    PheLeuLysIleIleAlaValTrpThrIleSerValGlyIleSerMet                              195200205                                                                     ProIleProValPheGlyLeuGlnAspAspS erLysValPheLysGlu                             210215220                                                                     GlySerCysLeuLeuAlaAspAspAsnPheValLeuIleGlySerPhe                              225230235 240                                                                 ValSerPhePheIleProLeuThrIleMetValIleThrTyrPheLeu                              245250255                                                                     ThrIleLysSerLeuGlnLysGlu AlaThrLeuCysValSerAspLeu                             260265270                                                                     GlyThrArgAlaLysLeuAlaSerPheSerPheLeuProGlnSerSer                              275 280285                                                                    LeuSerSerGluLysLeuPheGlnArgSerIleHisArgGluProGly                              290295300                                                                     SerTyrThrGlyArgArgThrMetGlnSer IleSerAsnGluGlnLys                             305310315320                                                                  AlaCysLysValLeuGlyIleValPhePheLeuPheValValMetTrp                              325 330335                                                                    CysProPhePheIleThrAsnIleMetAlaValIleCysLysGluSer                              340345350                                                                     CysAsnGluAspValIl eGlyAlaLeuLeuAsnValPheValTrpIle                             355360365                                                                     GlyTyrLeuSerSerAlaValAsnProLeuValTyrThrLeuPheAsn                              370 375380                                                                    LysThrTyrArgSerAlaPheSerArgTyrIleGlnCysGlnTyrLys                              385390395400                                                                  GluAsnLysLysProL euGlnLeuIleLeuValAsnThrIleProAla                             405410415                                                                     LeuAlaTyrLysSerSerGlnLeuGlnMetGlyGlnLysLysAsnSer                               420425430                                                                    LysGlnAspAlaLysThrThrAspAsnAspCysSerMetValAlaLeu                              435440445                                                                     GlyLysGlnHis SerGluGluAlaSerLysAspAsnSerAspGlyVal                             450455460                                                                     AsnGluLysValSerCysVal                                                         465470                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: oligonucleotide                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATGGTACACTGGCTGG GGGGTGGGCTGAGTTGACGGTGGCT42                                 (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: oligonucleotide                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TACCACGCGGCCAAGAGCCTTTACCA26                                                  (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: oligonucleotide                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TGGTGCTAGAGATCTGCTGACGTTC25                                                   ( 2) INFORMATION FOR SEQ ID NO:11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: oligonucleotide                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GAGAAGTC AGACACACAGAAAGTCTGTGTAAGTTTTACAACTTGC45                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OLIGONUCLEOTIDE                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT42                              

What is claimed is:
 1. An isolated nucleic acid molecule encoding ahuman 5-HT_(1E) receptor, having the amino acid sequence shown inFIG.
 1. 2. An isolated nucleic acid molecule of claim 1, wherein thenucleic acid molecule is a DNA molecule.
 3. An isolated nucleic acidmolecule of claim 2, wherein the DNA molecule is a cDNA molecule.
 4. Avector comprising the nucleic acid molecule of claim
 1. 5. A vector ofclaim 4 adapted for expression in a mammalian cell which comprises theregulatory elements necessary for expression of the nucleic acid in themammalian cell so located relative to the nucleic acid encoding the5-HT_(1E) receptor as to permit expression thereof.
 6. A plasmidcomprising the vector of claim
 5. 7. A mammalian cell comprising theplasmid of claim
 6. 8. A plasmid comprising the vector of claim
 4. 9. Athymidine kinase deficient L(Ltk⁻) cell comprising the plasmid of claim8.
 10. A Y1 cell comprising the plasmid of claim
 8. 11. A plasmid whichis pcEXV-hp75d (ATCC Accession No. 75138).
 12. An Ltk⁻ cell comprisingthe plasmid of claim 11 which is 5-HT_(1E) -7 (ATCC Accession No. CRL10913).
 13. A Y1 cell comprising the plasmid of claim 11 which isY-5-HT_(1E) (ATCC Accession No. CRL 10914).